System for simultaneous detection of multiple binding events in the same reaction

ABSTRACT

Methods and compositions are provided for detecting target molecules, e.g. DNA sequences, particularly single nucleotide polymorphisms, using a pair of nucleotide sequences, a primer and a snp detection sequence, where the snp detection sequence binds downstream from the primer to the target DNA in the direction of primer extension, or ligands and receptors. The methods employ e-tags comprising a mobility-identifying region joined to a detectable label and a target-binding region. The result of the binding of the target-binding region to the target is to have a bond cleaved in the starting material with the production of a detectable product with a different mobility from the starting material, where the different e-tags can be separated and detected

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/561,579, filed Apr. 28, 2000, which is a continuation-in-part ofapplication Ser. No. 09/303,029, filed Apr. 30, 1999, which disclosureis incorporated herein by reference.

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of this invention is electrophoretically separablecompositions for use in multiplex detection.

[0004] 2. Background of the Invention

[0005] As the human genome is elucidated, there will be numerousopportunities for performing assays to determine the presence ofspecific sequences, distinguishing between alleles in homozygotes andheterozygotes, determining the presence of mutations, evaluatingcellular expression patterns, etc. In many of these cases one will wishto determine in a single reaction, a number of different characteristicsof the same sample. Also, there will be an interest in determining thepresence of one or more pathogens, their antibiotic resistance genes,genetic subtype and the like.

[0006] In many assays, there will be an interest in determining thepresence of specific sequences, whether genomic, synthetic or cDNA.These sequences may be associated particularly with genes, regulatorysequences, repeats, multimeric regions, expression patterns, and thelike

[0007] There is and will continue to be comparisons of the sequences ofdifferent individuals. It is believed that there will be about onepolymorphism per 1,000 bases, so that one may anticipate that there willbe an extensive number of differences between individuals. By singlenucleotide polymorphism (snp's) is intended that there will be aprevalent nucleotide at the site, with one or more of the remainingbases being present in substantially smaller percent of the population.

[0008] For the most part, the snp's will be in non-coding regions,primarily between genes, but will also be present in exons and introns.In addition, the great proportion of the snp's will not affect thephenotype of the individual, but will clearly affect the genotype. Thesnp's have a number of properties of interest. Since the snp's will beinherited, individual snp's and/or snp patterns may be related togenetic defects, such as detections, insertions and mutations involvingone or more bases in genes. Rather than isolating and sequencing thetarget gene, it will be sufficient to identify the snp's involved.

[0009] In addition, the snp's may be used in forensic medicine toidentify individuals. While other genetic markers are available, thelarge number of snp's and their extensive distribution in thechromosomes, make the snp's an attractive target. Also, by determining aplurality of snp's associated with a specific phenotype, one may use thesnp pattern as an indication of the phenotype, rather than requiring adetermination of the genes associated with the phenotype.

[0010] The need to determine many analytes or nucleic acid sequences(for example multiple pathogens or multiple genes or multiple geneticvariants) in blood or other biological fluids has become increasinglyapparent in many branches of medicine. The need to study differentialexpression of multiple genes to determine toxicologically-relevantoutcomes or the need to screen transfused blood for viral contaminantswith high sensitivity is clearly evident.

[0011] Thus most multi-analyte assays or assays which detect multiplenucleic acid sequences involve mutiple steps, have poor sensitivity andpoor dynamic range (2 to 100-fold differences in concentration of theanalytes is determined) and some require sophisticated instrumentation.

[0012] Some of the known classical methods for multianalyte assaysinclude the following:

[0013] a. The use of two different radioisotope labels to distinguishtwo different analytes.

[0014] b. The use of two or more different fluorescent labels todistinguish two or more analytes.

[0015] c. The use of lanthamide chelates where both lifetime andwavelength are used to distinguish two or more analytes.

[0016] d. The use of fluorescent and chemiluminescent labels todistinguish two or more analytes.

[0017] e. The use of two different enzymes to distinguish two or moreanalytes.

[0018] f. The use of enzyme and acridinium esters to distinguish two ormore analytes.

[0019] g. Spatial resolution of different analytes, for example, onarrays to identify and quantify multiple analytes.

[0020] h. The use of acridinium ester labels where lifetime ordioxetanone formation is used to quantify two different viral targets.

[0021] Thus an assay that has higher sensitivity, large dynamic range(10³ to 10⁴-fold differences in target nucleic acids levels), greaterdegree of multiplexing, and fewer and more stable reagents wouldincrease the simplicity and reliability of multianalyte assays.

[0022] The need to identify and quantify a large number of bases orsequences potentially distributed over centimorgans of DNA offers amajor challenge. Any method should be accurate, reasonably economical inlimiting the amount of reagents required and providing for a singleassay, which allows for differentiation of the different snp's ordifferentiation and quantitation of multiple genes.

[0023] Finally, while nucleic acid sequences provide extreme diversityfor situations that may be of biological or other interest, there areother types of compounds, such as proteins in proteomics that may alsooffer opportunities for multiplexed determinations.

[0024] 3. Brief Description of the Related Art

[0025] Holland (Proc. Natl. Acad. Sci. USA (1991) 88:7276) disclosesthat the exonuclease activity of the thermostable enzyme Thermusaquaticus DNA polymerase in PCR amplification to generate specificdetectable signal concomitantly with amplification.

[0026] The TaqMan assay is discussed by Lee in Nucleic Acid Research(1993) 21:16 3761).

[0027] White (Trends Biotechnology (1996) 14(12):478-483) discusses theproblems of multiplexing in the Taqman assay.

[0028] Marino, Electrophoresis (1996) 17:1499 describeslow-stringency-sequence specific PCR (LSSP-PCR). A PCR amplifiedsequence is subjected to single primer amplification under conditions oflow stringency to produce a range of different length amplicons.Different patterns are obtained when there are differences in sequence.The patterns are unique to an individual and of possible value foridentity testing.

[0029] Single strand conformational polymorphism (SSCP) yields similarresults. In this method the PCR amplified DNA is denatured and sequencedependent conformations of the single strands are detected by theirdiffering rates of migration during gel electrophoresis. As withLSSP-PCR above, different patterns are obtained that signal differencesin sequence. However, neither LSSP-PCR nor SSCP gives specific sequenceinformation and both depend on the questionable assumption that any basethat is changed in a sequence will give rise to a conformational changethat can be detected.

[0030] Pastinen, Clin. Chem. (1996) 42:1391 amplifies the target DNA andimmobilizes the amplicons. Multiple primers are then allowed tohybridize to sites 3′ and contiguous to an SNP site of interest. Eachprimer has a different size that serves as a code. The hybridizedprimers are extended by one base using a fluorescently labeleddideoxynucleoside triphosphate. The size of each of the fluorescentproducts that is produced, determined by gel electrophoresis, indicatesthe sequence and, thus, the location of the SNP. The identity of thebase at the SNP site is defined by the triphosphate that is used. Asimilar approach is taken by Haff, Nucleic Acids Res. (1997) 25:3749except that the sizing is carried out by mass spectroscopy and thusavoids the need for a label. However, both methods have the seriouslimitation that screening for a large number of sites will requirelarge, very pure primers that can have troublesome secondary structuresand be very expensive to synthesize.

[0031] Hacia, Nat. Genet. (1996) 14:441 uses a high density array ofoligonucleotides. Labeled DNA samples were allowed to bind to 96,60020-base oligonucleotides and the binding patterns produced fromdifferent individuals were compared. The method is attractive in thatSNP's can be directly identified, but the cost of the arrays is high andnon-specific hybridization may confound the accuracy of the geneticinformation

[0032] Fan (1997, October 6-8, IBC, Annapolis Md.) has reported resultsof a large scale screening of human sequence-tagged sites. The accuracyof single nucleotide polymorphism screening was determined byconventional ABI resequencing.

[0033] Allele specific oligonucleotide hybridization along with massspectroscopy has been discussed by Ross in Anal. Chem. (1997) 69:4197.

[0034] Holland, et al., PNAS USA (1991) 88, 7276-7280, describes use ofDNA polymerase 5′-3′ exonuclease activity for detection of PCR products.

[0035] U.S. Pat. No. 5,807,682 describes probe compositions fordetecting a plurality of nucleic acid targets.

SUMMARY OF THE INVENTION

[0036] Systems are provided comprising libraries of compositions forlinking to or linked to assay reagents for performing simultaneousdeterminations in a single container. The systems combine entities thatcomprise e-tags (electrophoretic tags capable of being separatedelectrophoretically with the entities to which they are attached in aspecific determination) that include mobility-identifying regionscomprising a first functionality bonded to an assay reagent and a secondfunctionality bonded to or for bonding to a detectable label, with asample under conditions which produce an analyte-dependent detectablechange in the mobility of the entities, means for moving the modifiedentities to an electrophoretic device, and a data processor forprocessing the data from the electrophoretic device. Libraries areemployed comprising a plurality of e-tag containing compositions, wherethe e-tags are joined to assay reagents, a unit of an assay reagent orprovide a functionality for linking to an assay reagent, where thelinkage may be cleavable. The assays employ reagents for homogeneous (norequired separation step) or heterogeneous (a separation step required)protocols.

[0037] The libraries comprise entities comprising electrophoretic tagsthat are small molecules (molecular weight of 150 to 5,000), usuallyother than oligomers, which can be used in any measurement techniquethat permits identification by mass, e.g. mass spectrometry, and ormass/charge ratio, as in mobility in electrophoresis. Simple variationsin mass and/or mobility of the e-tag leads to generation of a library ofe-tags, that can then be used to detect a plurality of individual eventsassociated with different molecular species, generally related species.The e-tags are designed to be easily and rapidly separated, particularlyin free solution without the need for a polymeric separation media.Quantitation is achieved using internal controls. Enhanced separation ofthe e-tags comprising a nucleotide in electrophoresis is achieved bymodifying the tags with positively charged moieties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIGS. 1A, B and C depict the specific sequences of the snpdetection sequences for the two alleles, the optical characteristics ofthe fluorescent dyes, and the cleaved fragments from the snp detectionsequences, respectively;

[0039]FIGS. 2A and B depict the CE separation of the reaction productsof Allele 1 after 0 and 40 cycles. CE instrument: Beckman P/ACE/5000with LIF detection. BGE: 2.5% LLD 30, 7M urea, 1×TBE. Capillary: 100 μmi.d., 375 μm o.d., Lc=27 cm, Ld=6.9 cm. Detection; λ_(ex)=488 nm,λ_(em)=520 nm. Injection: 5 s at 2.0 kV. Field strength: 100V/cm at rt.Peaks: P=unreacted snp detection sequence, P′=snp detection sequenceproduct;

[0040]FIGS. 3A and B depict the CE separation of the reaction productsof Allele 1 after 0 and 40 cycles. Experimental conditions are the sameas FIG. 2, except for BGE composition; 2% LDD30, 1×TBE;

[0041]FIG. 4 is a graph of the CE separation of a 1:1 mixture of the 40cycles products of Alleles 1 and 2, with experimental conditions asdescribed for FIG. 2;

[0042]FIG. 5 is a graph of the CE separation of a 1:10 mixture of the 40cycles products of Alleles 1 and 2, with experimental conditions asdescribed for FIG. 2;

[0043]FIG. 6 is an electopherogram of e-tags, which involved aseparation involving a 1000-fold difference in concentration;

[0044]FIGS. 7a, 7 b are the electropherograms of the analysis of 5 snpsof the cystic fibrosis genes using multiplexed PCR and the subject e-tagprobes. FIG. 7c is the electropherogram of the analysis of single snpsand triple x snps for the cystic fibrosis genes using multiplexed PCRand the subject e-tag probes along with an agarose gel separation of thetriple x PCR;

[0045]FIG. 8 is an electropherogram of a separation of 9 negativelycharged e-tag probes.

[0046]FIGS. 9A and 9B are electropherograms of probes employing apenultimate thiophosphate linkage in the e-tag probes to discouragecleavage after the first phosphate linkage;

[0047]FIG. 10 is a cartoon of a system for performing multiplexeddeterminations using e-tags.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0048] A system is provided for the simultaneous multiplexeddetermination of a plurality of events employing electrophoresis todistinguish the events, comprising an electrophoretic device forelectrophoretic separation and detection, a container containing a firstset of first agents, referred to as “e-tags,” comprising differingmobility regions and a second reagent composition comprising at leastone active second agent, under conditions where said second agentmodifies at least one member of said first agent set resulting in achange of electrophoretic mobility of said at least one member toprovide a modified member retaining said mobility region, and transferof said at least one modified member to said electrophoretic device forseparation and detection of said at least one modified member. Theelectrophoretic device may be connected to a data processor forreceiving and processing data from the device, as well as operating theelectrophoretic device

[0049] The systems are based on having libraries available comprising aplurality of e-tags that comprise at least a plurality of differentmobility-identifying regions, so as to be separable in anelectrophoresis with the entities to which the mobility-identifyingregions are attached. The mobility-identifying regions are retained inthe product of the reaction, where the product is modified by the gainand/or loss of a group that changes the mass and may also change thecharge of the product, as compared to the starting material. In someinstances, the mobility-identifying region may be joined to atarget-binding region by a cleavable bond, so that themobility-identifying region is released for analysis subsequent to themodification of the target-binding region, e.g. complex formation.

[0050] The subject invention provides compositions and methods forimproved analysis of complex mixtures, where one is interested in thesimultaneous identification of a plurality of entities, such as nucleicacid or amino acid sequences, snps, alleles, mutations, proteins,haptens, protein family members, expression products, etc., analysis ofthe response of a plurality of entities to an agent that can affect themobility of the entities, and the like. Libraries of differentiablecompounds are provided, where the compounds comprise amobility-identifying region (including mass-identifying region) (“mir”),that provides for ready identification by electrophoresis or massspectrometry (differentiation by mobility in an electrical field ormagnetic field), by itself or in conjunction with a detectable label.Depending on the determination the product may also include one or morenucleotides or their equivalent, one or more amino acids or theirequivalent, a functionality resulting from the release of thetarget-binding region or a modified functionality as a result of theaction of an agent on the target-binding region.

[0051] The methodology involves employing detectable tags that can bedifferentiated by electrophoretic mobility or mass. The tags comprisemobility-identifying regions joined to a moiety that will undergo achange to produce a product. Depending on the nature of the change, thechange may involve a change in mass and/or charge of the mir, therelease of the mir from all or a portion of the target-binding region ormay provide for the ability to sequester the mir from the startingmaterial for preferential release of the mir. The differentiable tags,whether identified by electrophoresis or mass spectrometry, comprisingthe mir, with or without the detectable label and a portion of thetarget-binding region will be referred to as “e-tags.”

[0052] In addition, the subject invention employs a variety of reagentsystems, where a binding event results in a change in mobility of thee-tag. The binding event is between a target-binding region and atarget, and the reagent system recognizes this event and changes thenature of the e-tag containing target-binding region, so that themobility and/or mass of the product is different from the startingmaterial. The reagent system will frequently involve an enzyme and thereagent system may comprise the target. The effect of the reagent systemis to make or break a bond by physical, chemical or enzymatic means.Each of the products of the different e-tag containing target-bindingregions can be accurately detected, so as to determine the occurrence ofthe binding event.

[0053] The subject invention may be used for a variety of multiplexedanalyses involving the action of one or more agents on a plurality ofreagents comprising the mir and a target-binding region that undergoes achange as a result of a chemical reaction, resulting in a change inmobility of the product as compared to the starting material. Thereaction may be the result of addition or deletion in relation to thetarget-binding region, so that the resulting product may be sequesteredfrom the starting material. The subject systems find use in nucleic acidand protein analyses, reactions, particularly enzyme reactions, whereone or more enzymes are acting on a group of different potential oractual substrates, and the like.

[0054] The e-tags are a group of reagents having a mir that with theother regions to which the mir is attached during separation provide forunique identification of an entity of interest. The mir of the e-tagscan vary from a bond to about 100 atoms in a chain, usually not morethan about 60 atoms, more usually not more than about 30 atoms, wherethe atoms are carbon, oxygen, nitrogen, phosphorous, boron and sulfur.Generally, when other than a bond, the mir will have from 0 to 40, moreusually from 0 to 30 heteroatoms, which in addition to the heteroatomsindicated above will include halogen or other heteroatom. The totalnumber of atoms other than hydrogen will generally be fewer than 200atoms, usually fewer than 100 atoms. Where acid groups are present,depending upon the pH of the medium in which the mir is present, variouscations may be associated with the acid group. The acids may be organicor inorganic, including carboxyl, thionocarboxyl, thiocarboxyl,hydroxamic, phosphate, phosphite, phosphonate, sulfonate, sulfinate,boronic, nitric, nitrous, etc. For positive charges, substituents willinclude amino (includes ammonium), phosphonium, sulfonium, oxonium,etc., where substituents will generally be aliphatic of from about 1-6carbon atoms, the total number of carbon atoms per heteroatom, usuallybe less than about 12, usually less than about 9. The mir may be neutralor charged depending on the other regions to which the mir is attached,at least one of the regions having at least one charge. Neutral mirswill generally be polymethylene, halo- or polyhaloalkylene or aralkylene(a combination of aromatic—includes heterocyclcic—and aliphatic groups),where halogen will generally be fluorine, chlorine, bromine or iodine,polyethers, particularly, polyoxyalkylene, wherein alkyl is of from 2-3carbon atoms, polyesters, e.g. polyglycolide and polylactide,dendrimers, comprising ethers or thioethers, oligomers of addition andcondensation monomers, e.g. acrylates, diacids and diols, etc. The sidechains include amines, ammonium salts, hydroxyl groups, includingphenolic groups, carboxyl groups, esters, amides, phosphates,heterocycles, particularly nitrogen heterocycles, such as the nucleosidebases and the amino acid side chains, such as imidazole and quinoline,thioethers, thiols, or other groups of interest to change the mobilityof the e-tag. The mir may be a homooligomer or a heterooligomer, havingdifferent monomers of the same or different chemical characteristics,e.g., nucleotides and amino acids. Desirably neutral massdifferentiating groups will be combined with short charged sequences toprovide the mir.

[0055] The charged mirs will generally have only negative or positivecharges, although, one may have a combination of charges, particularlywhere a region to which the mir is attached is charged and the mir hasthe opposite charge. The mirs may have a single monomer that providesthe different functionalities for oligomerization and carry a charge ortwo monomers may be employed, generally two monomers. One may usesubstituted diols, where the substituents are charged and dibasic acids.Illustrative of such oligomers are the combination of diols or diamino,such as 2,3-dihydroxypropionic acid, 2,3-dihydroxysuccinic acid,2,3-diaminosuccinic acid, 2,4-dihydroxyglutaric acid, etc. The diols ordiamino compounds can be linked by dibasic acids, which dibasic acidsinclude the inorganic dibasic acids indicated above, as well as dibasicacids, such as oxalic acid, malonic acid, succinic acid, maleic acid,furmaric acid, carbonic acid, etc. Instead of using esters, one may useamides, where amino acids or diamines and diacids may be employed.Alternatively, one may link the hydroxyls or amines with alkylene orarylene groups.

[0056] By employing monomers that have substituents that provide forcharges or which may be modified to provide charges, one can provide formirs having the desired mass/charge ratio. For example, by using serineor threonine, one may modify the hydroxyl groups with phosphate toprovide negatively charged mirs. With arginine, lysine and histidine,one provides for positively charged mirs. Oligomerization may beperformed in conventional ways to provide the appropriately sized mir.The different mirs having different orders of oligomers, generallyhaving from 1 to 20 monomeric units, more usually about 1 to 12, where aunit intends a repetitive unit that may have from 1 to 2 differentmonomers. For the most part, oligomers will be used with other thannucleic acid target-binding regions. The polyfunctionality of themonomeric units provides for functionalities at the termini that may beused for conjugation to other moieties, so that one may use theavailable functionality for reaction to provide a differentfunctionality. For example, one may react a carboxyl group with anaminoethylthiol, to replace the carboxyl group with a thiolfunctionality for reaction with an activated olefin.

[0057] By using monomers that have 1-3 charges, one may employ a lownumber of monomers and provide for mobility variation with changes inmolecular weight. Of particular interest are polyolpolycarboxylic acidshaving from about two to four of each functionality, such as tartaricacid, 2,3-dihydroxyterephthalic acid, 3,4-dihydroxyphthalic acid,Δ⁵-tetrahydro-3,4-dihydroxyphthalic acid, etc. To provide for anadditional negative charge, these monomers may be oligomerized with adibasic acid, such as a phosphoric acid derivative to form the phosphatediester. Alternatively, the carboxylic acids could be used with adiamine to form a polyamide, while the hydroxyl groups could be used toform esters, such as phosphate esters, or ethers such as the ether ofglycolic acid, etc. To vary the mobility, various aliphatic groups ofdiffering molecular weight may be employed, such as polymethylenes,polyoxyalkylenes, polyhaloaliphatic or—aromatic groups, polyols, e.g.sugars, where the mobility will differ by at least about 0.01, moreusually at least about 0.02 and more usually at least about 0.5.Alternatively, the libraries may include oligopeptides for providing thecharge, particularly oligopeptides of from 2-6, usually 2-4 monomers,either positive charges resulting from lysine, arginine and histidine ornegative charges, resulting from aspartic and glutamic acid. Of course,one need not use naturally occurring amino acids, but unnatural orsynthetic amino acids, such as taurine, phosphate substituted serine orthreonine, S-α-succinylcysteine, co-oligomers of diamines and aminoacids, etc.

[0058] Where the e-tags are used for mass detection, as with massspectrometry, the e-tags need not be charged but merely differ in mass,since a charge will be imparted to the e-tags by the mass spectrometer.Thus, one could use the same or similar monomers, where thefunctionalities would be neutral or made neutral, such as esters andamides of carboxylic acids. Also, one may vary the e-tags by isotopicsubstitution, such as ²H, ¹⁸O, ¹⁴C, etc.

[0059] The libraries will ordinarily have at least about 5 members,usually at least about 10 members, and may have 100 members or more, forconvenience generally having about 50-75 members. Some members may becombined in a single container or be provided in individual containers,depending upon the region to which the mir is attached. The members ofthe library will be selected to provide clean separations inelectrophoresis, when capillary electrophoresis is the analyticalmethod. To that extent, mobilities will differ as described above, wherethe separations may be greater, the larger the larger the number ofmolecules in the band to be analyzed. Particularly, non-sieving mediamay be employed in the separation.

[0060] An e-tag will be a molecule, which is labeled with a directlydetectable label or can be made so by having a functionality that can beused for bonding to a detectable label, if such label is required fordetection. The e-tags will be differentiated by their electrophoreticmobility, usually their mass/charge ratio, to provide differentmobilities for each e-tag. Although in some instances the e-tags mayhave identical mass/charge ratios, such as oligonucleotides, but differin size or shape and therefore exhibit different electrophoreticmobilities under appropriate conditions. Therefore, the tags will beamenable to electrophoretic separation and detection, although othermethods of differentiating the tags may also find use. The e-tag may bejoined to any convenient site on the target binding reagent, withoutinterfering with the synthesis, release and binding of the e-tag labeledreagent. For nucleotides, the e-tag may be bound to a site on the base,either an annular carbon atom or a hydroxyl or amino substituent.

[0061] The e-tag may be linked by a stable bond or one, which may becleavable, thermally, photolytically or chemically. There is an interestin cleaving the e-tag from the target-binding region in situations wherecleavage of the target-binding region results in significant cleavage atother than the desired site of cleavage, resulting in satellite cleavageproducts, such as di- and higher oligonucleotides and this family ofproducts interferes with the separation and detection of the e-tags.However, rather than requiring an additional step in the identificationof the tags by releasing them from the base to which they are attached,one can modify the target binding sequence to minimize obtainingcleavage at other than the desired bond, for example, the ultimate orpenultimate phosphate link in a nucleic acid sequence. For immunoassaysinvolving specific binding members, bonding of the e-tag will usually bethrough a cleavable bond to a convenient functionality, such as carboxy,hydroxy, amino or thiol, particularly as associated with proteins,lipids and saccharides.

[0062] If present, the nature of the cleavable link resulting in releaseof the e-tag may be varied widely. Numerous linkages are available,which are thermally, photolytically or chemically labile. See, forexample, U.S. Pat. No. 5,721,099. Where detachment of the product fromall or a portion of the target-binding region is desired, there arenumerous functionalities and reactants, which may be used. Conveniently,ethers may be used, where substituted benzyl ether or derivativesthereof, e.g. benzhydryl ether, indanyl ether, etc. may be cleaved byacidic or mild reductive conditions. Alternatively, one may employbeta-elimination, where a mild base may serve to release the product.Acetals, including the thio analogs thereof, may be employed, where mildacid, particularly in the presence of a capturing carbonyl compound, mayserve. By combining formaldehyde, HCl and an alcohol moiety, anα-chloroether is formed. This may then be coupled with an hydroxyfunctionality to form the acetal. Various photolabile linkages may beemployed, such as o-nitrobenzyl, 7-nitroindanyl, 2-nitrobenzhydrylethers or esters, etc.

[0063] For a list of cleavable linkages, see, for example, Greene andWuts, Protective Groups in Organic Synthesis, 2^(nd) ed. Wiley, 1991.The versatility of the various systems that have been developed allowsfor broad variation in the conditions for attachment of the e-tagentities.

[0064] Various functionalities for cleavage are illustrated by: silylgroups being cleaved with fluoride, oxidation, acid, bromine orchlorine; o-nitrobenzyl with light; catechols with cerium salts; olefinswith ozone, permanganate or osmium tetroxide; sulfides with singletoxygen or enzyme catalyzed oxidative cleavage with hydrogen peroxide,where the resulting sulfone can undergo elimination; furans with oxygenor bromine in methanol; tertiary alcohols with acid; ketals and acetalswith acid; α- and β-substituted ethers and esters with base, where thesubstituent is an electron withdrawing group, e.g., sulfone, sulfoxide,ketone, etc., and the like

[0065] The mir will link the target-binding region and the detectablelabel molecule, usually a fluorescer, or a functionality, which may beused for linking to a detectable label molecule. By having differentfunctionalities, which may be individually bonded to a detectable labelmolecule, one enhances the opportunity for diversity of the e-tags.Using different fluorescers for joining to the differentfunctionalities, the different fluorescers can provide differences inlight emission and mass/charge ratios for the e-tags.

[0066] As discussed previously, the mir may be an oligomer, where themonomers may differ as to mass and charge. For convenience and economy,monomers will generally be commercially available, but if desired, theymay be originally synthesized. Monomers which are commercially availableand readily lend themselves to oligomerization include amino acids, bothnatural and synthetic, monosaccharides, both natural and synthetic,while other monomers include hydroxyacids, where the acids may beorganic or inorganic, e.g. carboxylic, phosphoric, boric, sulfonic,etc., and amino acids, where the acid is inorganic, and the like. Insome instances, nucleotides, natural or synthetic, may find use. Themonomers may be neutral, negatively charged or positively charged ormodified to be charged or neutral, e.g. sugars that are phosphorylated,amino acids that are acylated. Normally, the charges of the monomers inthe mir will be the same, so that in referring to the mass/charge ratio,it will be related to the same charge. Where the label has a differentcharge from the mir, this will be treated as if the number of charges isreduced by the number of charges on the mir. For natural amino acids,the positive charges may be obtained from lysine, arginine andhistidine, while the negative charges may be obtained from aspartic andglutamic acid. For nucleotides, the charges will be obtained from thephosphate and any substituents that may be present or introduced ontothe base. For sugars, sialic acid and uronic acids of the varioussugars, or substituted sugars may be employed.

[0067] The mir may be joined in any convenient manner to the unit of thetarget-binding region, such as the base of the nucleoside or the aminoacid of a protein. Various functionalities which may be used includealkylamine, amidine, thioamide, ether, urea, thiourea, guanidine, azo,thioether and carboxylate, sulfonate, and phosphate esters, amides andthioesters.

[0068] Besides the nature of the mir, as already indicated, diversitycan be achieved by the chemical and optical characteristics of thelabel, the use of energy transfer complexes, variation in the chemicalnature of the mir, which affects mobility, such as folding, interactionwith the solvent and ions in the solvent, and the like. As alreadysuggested, the mir will usually be an oligomer, where the mir may besynthesized on a support or produced by cloning or expression in anappropriate host. Conveniently, polypeptides can be produced where thereis only one cysteine or serine/threonine/tyrosine, aspartic/glutamicacid, or lysine/arginine/histidine, other than an end group, so thatthere is a unique functionality, which may be differentiallyfunctionalized. By using protective groups, one can distinguish a sidechain functionality from a terminal amino acid functionality. Also, byappropriate design, one may provide for preferential reaction betweenthe same functionalities present at different sites on the mir. Whetherone uses synthesis or cloning for preparation of oligopeptides, will toa substantial degree depend on the length of the mir.

[0069] The e-tag, which is detected, will comprise the mir, generally alabel, and optionally a portion of the target-binding region, all of thetarget-binding region when the target is an enzyme and thetarget-binding region is the substrate. Generally, the e-tag will have acharge/mass ratio in the range of about −0.0001 to 1, usually in therange of about −0.001 to about 0.5. Mobility is proportional toq/M^(2/3), where q is the charge on the molecule and M is the mass ofthe molecule. Desirably, the difference in mobility under the conditionsof the determination between the closest electrophoretic labels will beat least about 0.001, usually 0.002, more usually at least about 0.01,and may be 0.02 or more.

[0070] Depending upon the reagent to which the e-tag is attached, theremay be a single e-tag or a plurality of c-tags, generally ranging fromabout 1-100, more usually ranging from about 1-40, more particularlyranging from about 1-20. The number of e-tags bonded to a singletarget-binding region will depend upon the sensitivity required, thesolubility of the c-tag conjugate, the effect on the assay of aplurality of e-tags, and the like. For oligomers or polymers, such asnucleic acids and poly(amino acids), e.g. peptides and proteins, one mayhave one or a plurality of e-tags, while for synthetic or naturallyoccurring non-oligomeric compounds, usually there will be only 1-3, moreusually 1-2 e-tags.

[0071] The e-tag for use in electrophoresis may be represented by theformula:

R-L-T

[0072] wherein R is a label, particularly a fluorescer, L is a mir, abond or a linking group as described previously, where L and the regionsto which L is attached provide for the variation in mobility of thee-tags. T comprises a portion of the target-binding region, particularlya nucleoside base, purine or pyrimidine, and is the base, a nucleoside,nucleotide or nucleotide triphosphate, an amino acid, either naturallyoccurring or synthetic, or other functionality that may serve toparticipate in the synthesis of an oligomer, when T is retained, and isotherwise a functionality resulting from the cleavage between L, themir, and the target-binding region. L provides a major factor in thedifferences in mobility between the different e-tags, in combinationwith the label and any residual entity, which remain with the mir. L mayor may not include a cleavable bond, depending upon whether the terminalentity to which L is attached is to be retained or completely removed.

[0073] L has been substantially described as the mir and as indicatedpreviously may include charged groups, uncharged polar groups or benon-polar. The groups may be alkylene and substituted alkylenes,oxyalkylene and polyoxyalkylene, particularly alkylene of from 2 to 3carbon atoms, arylenes and substituted arylenes, polyamides, polyethers,polyalkylene amines, etc. Substituents may include heteroatoms, such ashalo, phosphorous, nitrogen, oxygen, sulfur, etc., where the substituentmay be halo, nitro, cyano, non-oxo-carbonyl, e.g. ester, acid and amide,oxo-carbonyl, e.g. aldehyde and keto, amidine, urea, urethane,guanidine, carbamyl, amino and substituted amino, particularly alkylsubstituted amino, azo, oxy, e.g. hydroxyl and ether, etc., where thesubstituents will generally be of from about 0 to 10 carbon atoms, whileL will generally be of from about 1 to 100 carbon atoms, more usually offrom about 1 to 60 carbon atoms and preferably about 1 to 36 carbonatoms. L will be joined to the label and the target-binding region byany convenient functionality, such as carboxy, amino, oxy, phospo, thio,iminoether, etc., where in many cases the label and the target-bindingregion will have a convenient functionality for linkage.

[0074] The number of heteroatoms in L is sufficient to impart thedesired charge to the label conjugate, usually from about 1 to about200, more usually from about 2 to 100, heteroatoms. The heteroatoms in Lmay be substituted with atoms other than hydrogen.

[0075] The charge-imparting moieties of L may be, for example, aminoacids, tetraalkylammonium, phosphonium, phosphate diesters, carboxylicacids, thioacids, sulfonic acids, sulfate groups, phosphate monoesters,and the like and combinations of one or more of the above. The number ofthe above components of L is such as to achieve the desired number ofdifferent charge-imparting moieties. The amino acids may be, forexample, lysine, aspartic acid, alanine, gamma-aminobutyric acid,glycine, β-alanine, cysteine, glutamic acid, homocysteine, P-alanine andthe like. The phosphate diesters include, for example, dimethylphosphate diester, ethylene glycol linked phosphate diester, and soforth. The thioacids include, by way of example, thioacetic acid,thiopropionic acid, thiobutyric acid and so forth. The carboxylic acidspreferably have from 1 to 30 carbon atoms, more preferably, from 2 to 15carbon atoms and preferably comprise one or more heteroatoms and may be,for example, acetic acid derivatives, formic acid derivatives, succinicacid derivatives, citric acid derivatives, phytic acid derivatives andthe like. In one embodiment of the present invention the labelconjugates having different charge to mass ratios may comprisefluorescent compounds, each of which are linked to molecules that imparta charge to the fluorescent compound conjugate. As indicated previously,desirably the linking group has an overall negative charge, preferablyhaving in the case of a plurality of groups, groups of the same charge,where the total charge may be reduced by having one or more oppositelycharged moiety.

[0076] Of particular interest for L is to have two sub-regions, a commoncharged sub-region, which will be common to a group of e-tags, and avarying uncharged, a non-polar or polar sub-region, that will vary themass/charge ratio. This permits ease of synthesis, provides forrelatively common chemical and physical properties and permits ease ofhandling. For negative charges, one may use dibasic acids that aresubstituted with functionalities that permit low orders ofoligomerization, such as hydroxy and amino, where amino will usually bepresent as neutral amide. These charge imparting groups provide aqueoussolubility and allow for various levels of hydrophobicity in the othersub-region. Thus the uncharged sub-region could employ substituteddihydroxybenzenes, diaminobenzenes, or aminophenols, with one or greaternumber of aromatic rings, fused or non-fused, where substituents may behalo, nitro, cyano, alkyl, etc., allowing for great variation inmolecular weight by using a common building block. Where the otherregions of the e-tag impart charge to the c-tag, L may be neutral.

[0077] In some instances, where release of the e-tag results in anavailable functionality that can be used to react with a detectablelabel, there will be no need for R to be a functionality. The release ofthe e-tag can provide an hydroxyl, amino, carboxy or thiol group, whereeach may serve as the site for conjugation to the detectable label. Tothe extent that the e-tag is released free of a component of thetarget-binding region, this opportunity will be present. In that case, Ris the unreactive (under the conditions of the conjugation) terminus ofL and T is a functionality for release of the e-tag that may be joinedto all or a portion of the target-binding region or may be available forbinding to all or a portion of the target-binding region.

[0078] Combinations of particular interest comprise a fluorescentcompound and a different amino acid or combinations thereof in the formof a peptide or combinations of amino acids and thioacids or othercarboxylic acids. Such compounds are represented by the formula:

R′-L′-T′

[0079] wherein R′ is a fluorescer, L′ is is an amino acid or a peptideor combinations of amino acids and thioacids or other carboxylic acidsand T′ is a functionality for linking to a nucleoside base or is anucleoside, nucleotide or nucleotide triphosphate or other moiety asdescribed above for T.

[0080] In one embodiment of the present invention, the charge-impartingmoiety is conveniently composed primarily of amino acids but also mayinclude thioacids and other carboxylic acids having from one to fivecarbon atoms. The charge-imparting moiety may have from 1 to 30,preferably 1 to 20, more preferably, 1 to 10 amino acids per moiety andmay also comprise 1 to 3 thioacids or other carboxylic acids. Howeever,when used with an uncharged sub-region, the charged sub-region willgenerally have from 1-4, frequently 1-3 amino acids. As mentioned above,any amino acid, both naturally occurring and synthetic may be employed.

[0081] In a particular embodiment the label conjugates may berepresented by the formula:

Fluorescer-L″-(amino acid)_(n)-T″

[0082] wherein L″ is a bond or a linking group of from 1 to 20 atomsother than hydrogen, n is 1 to 20, and T″ comprises a nucleoside base,purine or pyrimidine, including a base, a nucleoside, a nucleotide ornucleotide triphosphates, an amino acid, or functionality for linking tothe target-binding region. An example of label conjugates in thisembodiment, by way of illustration and not limitation, is one in whichthe fluorescer is fluorescein, L″ is a bond in the form of an amidelinkage involving the meta-carboxyl of the fluorescein and the terminalamine group of lysine, and T″ is a nucleotide triphosphate. These labelconjugates may be represented as follows:

Fluorescein-(CO)NH—CH(CH₂)₃CH(NH₂)(amino acid)_(n)COX″

[0083] wherein X is as set forth in Table 1. TABLE 1 No. X Charge 1 OH−2 2 NH-lysine −1 3 NH-(lysine)₂ neutral 4 NH-alanine −3 5 NH-asparticacid −4 6 NH-(aspartic acid)₂ −5 7 NH-(aspartic acid)₃ −6 8 NH-(asparticacid)₄ −7 9 NH-(aspartic acid)₅ −8 10 NH-(aspartic acid)₆ −9 11NH-(aspartic acid)₇ −10 12 NH-alanine-lysine −2 (unique q/M) 13NH-aspartic acid-lysine −3 (unique q/M) 14 NH-(aspartic acid)₂-lysine −4(unique q/M) 15 NH-(aspartic acid)₃-lysine −5 (unique q/M) 16NH-(aspartic acid)₄-lysine −6 (unique q/M) 17 NH-(aspartic acid)₅-lysine−7 (unique q/M) 18 NH-(aspartic acid)₆-lysine −8 (unique q/M) 19NH-(aspartic acid)₇-lysine −9 (unique q/M) 20 NH-(aspartic acid)₈-lysine−10 (unique q/M) 21 NH-(lysine)₄ +1 22 NH-(lysine)₅ +2

[0084] wherein q is charge, M is mass and mobility is q/M^(2/3).Examples of such label conjugates are shown in FIG. 1C.

[0085] Table 2 shows various characteristics for the label conjugates.TABLE 2 No. Mass(M) Charge(g) M^(2/3) q/M^(2/3) Mobility 1 744.82 082.16765 0 0 2 877.02 0 91.62336 0 0 3 828.71 −1 88.22704 −0.01133−0.16546 4 970.71 −1 98.03767 −0.0102 −0.1489 5 700.82 −2 78.89891−0.02535 −0.37004 6 842.83 −2 89.22639 −0.2241 −0.32721 7 815.92 −387.31692 −0.03436 −0.50155 8 957.92 −3 97.17461 −0.03087 −0.45067 9931.02 −4 95.34677 −0.04195 −0.61242 10 1073.02 −4 104.8106 −0.03816−0.55712 11 1046 −5 103.0436 −0.04852 −0.70834 12 1188 −5 112.1702−0.04458 −0.65071 13 1161 −6 110.4642 −0.05432 −0.79291 14 1303 −6119.297 −0.05029 −0.7342 15 1276 −7 117.6433 −0.0595 −0.86861 16 1418 −7126.2169 −0.05546 −0.80961 17 1391 −8 124.6096 −0.0642 −0.9372 18 1533−8 132.952 −0.06017 −0.87839 19 1506 −9 131.3863 −0.0685 −0.99997 201648 −9 139.6205 −0.06451 −0.94167 21 793.52 1 85.7114 0.011667 0.17031622 935.52 1 95.65376 0.010454 0.152613

[0086] Another group of e-tags has a mir which is dependent on using analkylene or aralkylene (comprising a divalent aliphatic group having 1-2aliphatic regions and 1-2 aromatic regions, generally benzene), wherethe groups may be substituted or unsubstituted, usually unsubstituted,of from 2-16, more usually 2-12, carbon atoms, where the mir may linkthe same or different fluorescers to a monomeric unit, e.g. anucleotide. The mir may terminate in a carboxy, hydroxy or amino group,being present as an ester or amide. By varying the substituents on thefluorophor, one can vary the mass in units of at least 5 or more,usually at least about 9, so as to be able to obtain satisfactoryseparation in capillary electrophoresis. To provide further variation, athiosuccinimide group may be employed to join alkylene or aralkylenegroups at the nitrogen and sulfur, so that the total number of carbonatoms may be in the range of about 2-30, more usually 2-20. Instead ofor in combination with the above groups and to add hydrophilicity, onemay use alkyleneoxy groups.

[0087] The label conjugates may be prepared utilizing conjugatingtechniques that are well known in the art. The charge-imparting moiety Lmay be synthesized from smaller molecules that have functional groupsthat provide for linking of the molecules to one another, usually in alinear chain. Such functional groups include carboxylic acids, amines,and hydroxy- or thiol-groups. In accordance with the present inventionthe charge-imparting moiety may have one or more side groups pendingfrom the core chain. The side groups have a functionality to provide forlinking to a label or to another molecule of the charge-impartingmoiety.

[0088] Common functionalities of L resulting from the reaction of thefunctional groups employed are exemplified by forming a covalent bondbetween the molecules to be conjugated. Such functionalities aredisulfide, amide, thioamide, dithiol, ether, urea, thiourea, guanidine,azo, thioether, carboxylate and esters and amides containing sulfur andphosphorus such as, e.g. sulfonate, phosphate esters, sulfonamides,thioesters, etc., and the like.

[0089] The chemistry for performing the types of syntheses to form thecharge-imparting moiety as a peptide chain is well known in the art.See, for example, Marglin, et al., Ann. Rev. Biochem. (1970) 39:841-866.In general, such syntheses involve blocking, with an appropriateprotecting group, those functional groups that are not to be involved inthe reaction. The free functional groups are then reacted to form thedesired linkages. The peptide can be produced on a resin as in theMerrifield synthesis (Merrifield, J. Am. Chem. Soc. (1980) 85:2149-2154and Houghten et al., Int. J. Pep. Prot. Res. (1980) 16:311-320. Thepeptide is then removed from the resin according to known techniques.

[0090] A summary of the many techniques available for the synthesis ofpeptides may be found in J. M. Stewart, et al., “Solid Phase PeptideSynthesis, W. H. Freeman Co, San Francisco (1969); and J. Meienhofer,“Hormonal Proteins and Peptides”, (1973), vol. 2, p 46., Academic Press(New York), for solid phase peptide synthesis and E. Schroder, et al.,“The Peptides, vol. 1, Academic Press (New York), 1965 for solutionsynthesis.

[0091] In general, these methods comprise the sequential addition of oneor more amino acids, or suitably protected amino acids, to a growingpeptide chain. Normally, a suitable protecting group protects either theamino or carboxyl group of the first amino acid. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the final peptide.The protecting groups are removed, as desired, according to knownmethods depending on the particular protecting group utilized. Forexample, the protecting group may be removed by reduction with hydrogenand palladium on charcoal, sodium in liquid ammonia, etc.; hydrolysiswith trifluoroacetic acid, hydrofluoric acid, and the like.

[0092] After the synthesis of the peptide is complete, the peptide isremoved from the resin by conventional means such as ammonolysis,acidolysis and the like. The fully deprotected peptide may then bepurified by techniques known in the art such as chromatography, forexample, adsorption chromatography; ion exchange chromatography,partition chromatography, high performance liquid chromatography, thinlayer chromatography, and so forth.

[0093] As can be seen, the selected peptide representing acharge-imparting moiety may be synthesized separately and then attachedto the label either directly or by means of a linking group. On theother hand, the peptide may be synthesized as a growing chain on thelabel. In any of the above approaches, the linking of the peptide oramino acid to the label may be carried out using one or more of thetechniques described above for the synthesis of peptides or for linkingmoieties to labels.

[0094] Synthesis of e-tags comprising nucleotides can be easily andeffectively achieved via assembly on solid phase support during probesynthesis using standard phosphoramidite chemistries. The e-tags areassembled at the 5 end of probes after coupling of a final nucleosidicresidue, which becomes part of the e-tag during the assay. In oneapproach, the e-tag is constructed sequentially from a single or severalmonomeric phosphoramidite building blocks (one containing a dyeresidue), which are chosen to generate tags with unique electrophoreticmobilities based on their mass to charge ratio. The e-tag is thuscomposed of monomeric units of variable charge to mass ratios bridged byphosphate linkers (Figure A). The separation of e-tags, which differ by9 mass units (Table 3) has been demonstrated. The nucleosidicphosphoramidites employed for tag

[0095] synthesis are initially either modified or natural residues.Fluorescein has been the initial dye employed but other dyes can be usedas well. Some of the combinations of phosphoramidite building blockswith their predicted elution times are presented in Table 4. e-tags aresynthesized to generate a contiguous spectrum of signals, one elutingafter another with none of them coeluting (Figure B).

TABLE 3 e-tags that have been separated on a LabCard (detection: 4.7 cm;V/cm). Elution Time E-Tag on CE (sec) Mass

385 778

428 925

438 901

462 994

480 985

555 961

[0096] TABLE 4 Predicted and experimental (*) elution times of e-tags.C₃, C₆, C₉, C₁₈, are commercially available phosphoramidite spacers fromGlen Research, Sterling VA. The units are derivatives ofN,N-diisopropyl, O-cyanoethyl phosphoramidite, which in the followingformulas will be indicated by “Q”. C₃ is DMT (dimethoxytrityl)oxypropylQ; C₆ is DMToxyhexyl Q; C₉ is DMToxy(triethyleneoxy) Q; C₁₂ isDMToxydodecyl Q; C₁₈ is DMToxy(hexaethyleneoxy) Q. Etag Charge ElutionTime

−9 41.12

−8 43.72

−9 45.66

−8 48.14

−7 51.21

−6 53.53

−6 55.13

−5 57.66

−5 60.00

−5 62.86

−6 65.00*

−5 67.50*

−4 69.61

−4 72.00*

[0097] All of the above e-tags work well and are easily separable andelute after 40 minutes. To generate tags that elute faster, highlycharged low molecular weight tags are required. Several types ofphosphoramidite monomers allow for the synthesis of highly charged tagswith early elution times. Use of dicarboxylate phosphoramidites (FIG.5A) allows for the addition of 3 negative charges per coupling ofmonomer. A variety of fluorescein derivatives (FIG. 5B) allow the dyecomponent of the tag to carry a higher mass than standard fluorescein.Polyhydroxylated phosphoramidites (FIG. 6) in combination with a commonphosphorylation reagent enable the synthesis of highly phosphorylatedtags. Combinations of these reagents with other mass modifier linkerphosphoramidites allow for the synthesis of tags with early elutiontimes.

[0098] The aforementioned label conjugates with differentelectrophoretic mobility permit a multiplexed amplification anddetection of multiple targets, e.g. nucleic acid targets. The labelconjugates are linked to oligonucleotides in a manner similar to thatfor labels in general, by means of linkages that are enzymaticallycleavable. It is, of course, within the purview of the present inventionto prepare any number of label conjugates for performing multiplexeddeterminations. Accordingly, for example, with 40 to 50 different labelconjugates separated in a single separation channel and 96 differentamplification reactions with 96 separation channels on a single plasticchip, one can detect 4000 to 5000 single nucleotide polymorphisms.

[0099] The separation of e-tags, which differ by 9 mass units (Table 3)has been demonstrated as shown in FIG. 7. The penultimate couplingduring probe synthesis is initially carried out using commerciallyavailable modified (and unmodified) phosphoramidites (Table 4). Thisresidue is able to form hydrogen bonds to its partner in the targetstrand and is considered a mass modifier but could potentially be acharge modifier as well. The phosphate bridge formed during thiscoupling is the linkage severed during a 5′-nuclease assay. The finalcoupling is done using a phosphoramidite analogue of a dye. Fluoresceinis conveniently employed, but other dyes can be used as well.

[0100] One synthetic approach is outlined in Scheme 1. Starting withcommercially available 6-carboxy fluorescein, the phenolic hydroxylgroups are protected using an anhydride. Isobutyric anhydride inpyridine was employed but other variants are equally suitable. It isimportant to note the significance of choosing an ester functionality asthe protecting group. This species remains intact though thephosphoramidite monomer synthesis as well as during oligonucleotideconstruction. These groups are not removed until the synthesized oligois deprotected using ammonia. After protection the crude material isthen activated in situ via formation of an N-hydroxy succinimide ester(NHS-ester) using DCC as a coupling agent. The DCU byproduct is filteredaway and an amino alcohol is added. Many amino alcohols are commerciallyavailable some of which are derived from reduction of amino acids. Onlythe amine is reactive enough to displace N-hydroxy succinimide.

[0101] Upon standard extractive workup, a 95% yield of product isobtained. This material is phosphitylated to generate thephosphoramidite monomer (Scheme 1). For the synthesis of additionale-tags, a symmetrical bisamino alcohol linker is used as the aminoalcohol (Scheme 2). As such the second amine is then coupled with amultitude of carboxylic acid derivatives (Table 3) prior to thephosphitylation reaction. Using this methodology hundreds if notthousands of e-tags with varying charge to mass ratios can easily beassembled during probe synthesis on a DNA synthesizer using standardchemistries.

[0102] Additional e-tags are accessed via an alternative strategy whichuses 5-aminofluorescein as starting material (Scheme 3). Addition of5-aminofluorescein to a great excess of a diacid chloride in a largevolume of solvent allows for the predominant formation of themonoacylated product over dimer formation. The phenolic groups are notreactive under these conditions. Aqueous workup converts the terminalacid chloride to a carboxylic acid. This product is analogous to6-carboxy fluorescein and using the same series of steps is converted toits protected phosphoramidite monomer (Scheme 3). There are manycommercially available di(acid chorides) and diacids, which can beconverted to diacid chlorides using SOCl₂ or acetyl chloride. Thismethodology is highly attractive in that a second mass modifier is used.As such, if one has access to 10 commercial modified phosphoramiditesand 10 diacid chlorides and 10 amino alcohols there is a potential for1000 different e-tags. There are many commercial diacid chlorides andamino alcohols (Table 6). These synthetic approaches are ideally suitedfor combinatorial chemistry.

TABLE 5 Benzoic acid derivatives as mass and charge modifiers. (Mass iswritten below each modifier)

[0103] TABLE 6 Mass and charge modifiers that can be used for conversionof amino dyes into e-tag phosphoramidite monomers.

[0104] A variety of maleimide derivatized e-tags have also beensynthesized. These compounds were subsequently bioconjugated to 5′-thioladorned DNA sequences and subjected to the 5′-nuclease assay. Thespecies formed upon cleavage are depicted in Table 7. TABLE 7 E-tagsderived from maleimide linked precursors.

[0105] As a matter of convenience, predetermined amounts of reagentsemployed in the present invention can be provided in a kit in packagedcombination. A kit can comprise in packaged combination a target-bindingregion, e.g. oligonucleotide primer for each polynucleotide suspected ofbeing in said set wherein each of said primers is hybridizable to afirst sequence of a respective polynucleotide if present, a templatedependent polynucleotide polymerase, nucleoside triphosphates, and a setof oligonucleotide snp detection sequences, each of said oligonucleotideprobes having a fluorescent label at its 5′-end and having a sequence atits 5′-end that is hybridizable to a respective polynucleotide whereineach of said labels is cleavable from said oligonucleotide probe.Alternatively, the target-binding region may be an antibody fordetecting ligands or enzyme substrate for detecting enzymes.

[0106] The kit may further comprise a device for conducting capillaryelectrophoresis. For nucleic acid determinations, the e-tag isreleasable by a template dependent polynucleotide polymerase having 5′to 3′ exonuclease activity. The kit can further include various bufferedmedia, some of which may contain one or more of the above reagents.

[0107] The relative amounts of the various reagents in the kits can bevaried widely to provide for concentrations of the reagents necessary toachieve the objects of the present invention. Under appropriatecircumstances one or more of the reagents in the kit can be provided asa dry powder, usually lyophilized, including excipients, which ondissolution will provide for a reagent solution having the appropriateconcentrations for performing a method or assay in accordance with thepresent invention. Each reagent can be packaged in separate containersor some reagents can be combined in one container where cross-reactivityand shelf life permit. The kits may also include a written descriptionof a method in accordance with the present invention as described above.

[0108] In one embodiment of the kit, the e-tags are fluorescentconjugates represented by the formula:

R-L-T

[0109] wherein R is a fluorescer, L is a mir, as described previously,and T is a functionality for binding to a nucleoside base, purine orpyrimidine, or a nucleoside base, a nucleoside, nucleotide or nucleotidetriphosphates, or other member of the target-binding region.

[0110] In another embodiment of a kit, the e-tags are fluorescentconjugates represented by the formula:

R′-L′-T′

[0111] wherein R′ is a fluorescer, L′ is a bond, a combination of aneutral sub-region and a charged sub-region, an amino acid or a peptideor combinations of amino acids and thioacids or other carboxylic acidsand T′ is a nucleotide, nucleotide triphosphates or functionality forbinding to a member of the target-binding region.

[0112] In another embodiment of a kit, the e-tag is a fluorescentconjugate represented by the formula:

Fluorescer-L″-(amino acid)_(n)

[0113] wherein L″ with (amino acid)_(n) is a mir, where L″ is a bond ora linking group of from 1 to 20 atoms in the chain and n is 1 to 100,usually 1 to 20, more usually 1 to 10. The fluorescer may befluorescein, the amino acid may be lysine and L″ may be a bond in theform of an amide linkage involving the meta-carboxyl of the fluoresceinand the terminal amine group of lysine.

[0114] In another embodiment of a kit in accordance with the invention,the e-tag is a label conjugate represented by the formula:

Fluorescein-(CO)NH—CH(CH₂)₃CH(NH₂)COX

[0115] wherein X is selected from the group consisting of: OH,NH-lysine, NH-(lysine)₂, NH-alanine, NH-aspartic acid, NH-(asparticacid)₂, NH-(aspartic acid)₃, NH-(aspartic acid)₄, NH-(aspartic acid)₅,NH-(aspartic acid)₆, NH-(aspartic acid)₇, NH-alanine-lysine, NH-asparticacid-lysine, NH-(aspartic acid)₂-lysine, NH-(aspartic acid)₃-lysine,NH-(aspartic acid)₄-lysine, NH-(aspartic acid)₅-lysine, NH-(asparticacid)₆-lysine, NH-(aspartic acid)₇-lysine, NH-(aspartic acid)₈-lysine,NH-(lysine)₄, and NH-(lysine)₅.

[0116] The kits will usually have at least about 5 different e-tags forconjugation, more usually at least about 10, frequently at least about25 and may have 50 or more, usually not more than about 1,000. Thee-tags will differ as to mobility, including mass/charge ratio andnature of charge, e.g. overall positive or negative, detectable moiety,e.g. fluorophore, electrochemical, etc, or functionality for linking adetectable moiety, e.g. maleimide, mercaptan, aldehyde, ketone, etc.

[0117] The e-tags described above may terminate in an appropriatefunctionality for linking to a nucleotide, nucleotide triphosphate orother molecule of interest or may terminate in such moieties.

[0118] The methodologies that may be employed involve heterogeneous andhomogeneous techniques, where heterogeneous normally involves aseparation step, where unbound label is separated from bound label,where homogeneous assays do not require, but may employ a separationstep. One group of assays will involve nucleic acid detection, whichincludes sequence recognition, snp detection and scoring, transcriptionanalysis, allele determinations, HLA determinations, or otherdetermination associated with variations in sequence. The use of thedetermination may be forensic, mRNA determinations, mutationdeterminations, allele determinations, MHC determinations, haplotypedeterminations, single nucleotide polymorphism determinations, etc. Themethodology may include assays dependent on 5′-nuclease activity, as inthe use of the polymerase chain reaction or in Invader technology,3′-nuclease activity, restriction enzymes and ribonuclease H, all ofthese methods involving catalytic cleavage of a phosphate linkage, whereone to two oligonucleotides are bound to the target template.Alternatively, one may use channeling, where first and second agents arebound to first and second oligonucleotides, which bind proximally to thesame target nucleic acid template. By having a label generating amediator active in the cleavage of a bond present in the second agent towhich an e-tag is linked, the e-tag will be released only when the twoagents are proximally bound to the target template. The mediator may bephysical, e.g. electromagnetic radiation or chemical, e.g. singletoxygen or hydrogen peroxide. By providing for release of the agent towhich the e-tag is bonded from the template, one can amplify the numberof e-tags for a single target. Alternatively, one may have a pluralityof e-tags that are bonded to the agent bound to the target, wherebinding to the target permits separation from e-tag labeled agent thatis unbound. The e-tags bound to the target may then be releasedproviding for a plurality of e-tags for a single target.

[0119] Instead of nucleic acid pairing, one may employ specific bindingmember pairing. There are a large number of specific binding pairsassociated with receptors, such as antibodies, poly- and monoclonal,enzymes, surface membrane receptors, lectins, etc., and ligands for thereceptors, which may be naturally occurring or synthetic molecules,protein or non-protein, such as drugs, hormones, enzymes, ligands, etc.The specific binding pair has many similarities to the binding ofhomologous nucleic acids, significant differences being that onenormally cannot cycle between the target and the agent and one does nothave convenient phosphate bonds to cleave. For heterogeneous assays, thebinding of the specific binding pair is employed to separate the boundfrom the unbound e-tag bonded agents, while with homogeneous assays, theproximity of the specific binding pairs allow for release of the c-tagsfrom the complex.

[0120] For an inclusive but not exclusive listing of the various mannersin which the subject invention may be used, the following table isprovided.

[0121] Recognition Event Leads to Generation or Modification of E-Tags.Recognition Event e-tag Activation Amplification Mode Format BindingAssays (solution Phase Multiplexed assays (2-1000) e-tag generationfollowed by leading to release of library of e- separation by CE, HPLCor Mass tags. Every e-tag codes for a Spectra) unique binding event orassay. Hybridization followed by 5′ Nuclease assay PCR, Invader Sequencerecognition for enzyme recognition example for multiplexed geneexpression, SNP's scoring etc . . . 3′ Nuclease assay Multiplexed assaysSequence recognition Restriction enzymes Multiplexed assays Sequencerecognition Ribonuclease H Multiplexed assays Sequence recognitionHybridization followed by Singlet Oxygen Single e-tag release perbinding Multiplexed assays Sequence channeling event recognitionHybridization followed by Singlet Oxygen Amplification due to turnoverof e- Multiplexed assays Sequence channeling tag binding moietyrecognition Amplification due to release of Multiplexed assays Sequencemultiple e-tags (10 to 100,000) per recognition binding event Hydrogenperoxide Amplification due to turnover of e- Multiplexed assays Sequencetag binding moiety recognition Amplification due to release ofMultiplexed assays Sequence multiple e-tags (10 to 100,000) perrecognition binding event Light; Energy Transfer Amplification due toturnover Multiplexed assays Sequence (Photocleavage) of e-tag bindingrecognition moiety Amplification due to release of Multiplexed assaysSequence multiple e-tags (10 to 100,000) per recognition binding eventIMMUNO-ASSYS Sandwich assays Singlet Oxygen A few (2-10) e-tags releaseper Proteomics Antibody-1 decorated with binding event MultiplexedImmunoassays Sensitizer while antibody-2 Is decorated with singletoxygen cleavable e-tags Singlet Oxygen Amplification due to release ofProteomics multiple e-tags (10 to 100,000) per Multiplexed Immunoassaysbinding event Sandwich assays Hydrogen Peroxide A few (2-10) e-tagsrelease per Proteomics Antibody-1 decorated with binding eventMultiplexed Immunoassays Glucose oxidase while antibody-2 Is decoratedwith hydrogen peroxide cleavable e-tags Hydrogen Peroxide Amplificationdue to release of Proteomics multiple e-tags (10 to 100,000) perMultiplexed Immunoassays binding event Competition assays Singlet OxygenA few (2-10) e-tags release per Antibody-1 decorated with binding eventSensitizer while Antigen Is decorated with singlet oxygen cleavablee-tags Singlet Oxygen Amplification due to release of multiple e-tags(10 to 100,000) per binding event Competition assays Antibody-1decorated with Glucose oxidase while antigen Is decorated with hydrogenperoxide cleavable e-tags Hydrogen Peroxide A few (2-10) e-tags releaseper binding event Hydrogen Peroxide Amplification due to release ofmultiple e-tags (10 to 100,000) per binding event Binding Assays (SolidPhase e- Multiplexed assays (2-1000) tag generation followed by leadingto release of library of e- separation by CE, HPLC or Mass tags. Everye-tag codes for a Spectra) unique binding event or assay. HybridizationLight; Enzymes, As an alternative to Branched chain Sequence recognitionfor Capture of Target on solid Singlet oxygen, Hydrogen assay; Digene'sRNA:DNA duplex; example for gene expression, Surface. A number of e-tagPeroxide Fluoride, High Sensitivity sequence SNP's scoring; Pathogenlabeled probes are hybridized to Reducing agents, Mass identificationassay. detection; etc . . . the target. Unhybridized e-tag SpectraOthers Amplification due to release of Can be carried out on Patches inlabeled probes are removed. E-tag multiple e-tags (10 to 100,000) perMicrofluidic channels— is released and separated and binding event³Integrated assay and separation identified. device Immunoassays Sandwichassays Light; Enzymes, A few (2-10) e-tags release per ProteomicsAntibody-1 is attached to a solid Singlet oxygen, Hydrogen binding eventMultiplexed Immunoassays surface while antibody-2 Peroxide Fluoride,Amplification due to release of Can be carried out on Patches in isdecorated with cleavable e-tags Reducing agents, Mass Multiple e-tags(10 to 100,000) per Microfluidic channels— Spectra Others binding eventIntegrated assay and separation device Competition assays Light;Enzymes, A few (2-10) e-tags release per Proteomics Antibody-1 isattached to solid Singlet oxygen, Hydrogen binding event MultiplexedImmunoassays surface while Antigen Peroxide Fluoride, Amplification dueto release of Can be carried out on Patches in Is decorated withcleavable e-tags Reducing agents, Mass multiple e-tags (10 to 100,000)per Microfluidic channels— Spectra Others binding event Integrated assayand separation device

[0122] As indicated in the table, for amplification one may use thermalcycling. The cleavage of the nucleic acid bound to the template resultsin a change in the melting temperature of the e-tag residue with releaseof the e-tag. By appropriate choice of the primer and/or protocol, onecan retain the primer bound to the template and the e-tag containingsequence can be cleaved and released from the template to be replaced byan e-tag containing probe.

[0123] In determinations involving nucleic acids, since snp detectionis, for the most part, the most stringent in its requirements, most ofthe description will be directed toward the multiplexed detection ofsnps. For other nucleic acid analyses, frequently the protocols will besubstantially the same, although in some instances somewhat differentprotocols will be employed for snps, because of the greater demands snpsmake on fidelity. For proteins, the protocols will be substantiallydifferent and will be described independently of the snp protocols.

[0124] As exemplary of the subject invention, four targetpolynucleotides T1, T2, T3 and T4 are employed. Oligonucleotide primersPR1, PR2, PR3 and PR4 are employed, each respectively capable ofhybridizing to a sequence in the respective target polynucleotides. Alsoemployed are four oligonucleotide snp detection sequences, PB1, PB2, PB3and PB4. Each of the snp detection sequences comprises a fluorescentlabel F1, F2, F3 and F4, respectively. In this example, there is amismatch between PB2 and T2, which comprises a single nucleotidepolymorphism. The reaction medium comprising the above reagents andnucleoside triphosphates and a template dependent polynucleotidepolymerase having 5′ to 3′ exonuclease activity is treated underamplification conditions. Primers PR1, PR2, PR3 and PR4 hybridize totheir respective target polynucleotides and are extended to yieldextended primers EPR1, EPR2, EPR3 and EPR4. snp detection sequences PB1,PB3 and PB4, which hybridize with their respective targetpolynucleotides, are acted upon by the exonuclease to cleave a singlenucleotide bearing the respective fluorescent label. PB2, which does notbind to the target polynucleotide, is not cleaved. Cleaved fragments F1,F3 and F4 are injected into a separation channel in a chip forconducting electroseparation. The labels are identified by theirspecific mobility and fluorescence upon irradiation. The separatedlabels are related to the presence and amount of the respective targetpolynucleotide.

[0125] One, usually a plurality, of snp's, is simultaneously determinedby combining target DNA with one or a plurality, respectively, ofreagent pairs under conditions of primer extension. Each pair ofreagents includes a primer which binds to target DNA and a snp detectionsequence, normally labeled, which binds to the site of the snp and hasan e-tag, usually at its 5′-end and the base complementary to the snp,usually at other than a terminus of the snp detection sequence. Theconditions of primer extension employ a polymerase having 5′-3′exonuclease activity, dNTP's and auxiliary reagents to permit efficientprimer extension. The primer extension is performed, whereby detectorsequences bound to the target DNA are degraded with release of thee-tag. By having each snp associated with its own e-tag, one candetermine the snp's, which are present in the target DNA for which pairsof reagents have been provided.

[0126] The pairs of reagents are DNA sequences, which are related to asnp site. The primer binds to the target DNA upstream from the snp sitein the direction of extension. The labeled detector sequence bindsdownstream from the primer in the direction of extension and binds to asequence, which includes the snp. The primer sequence will usually be atleast about 12 bases long, more usually at least 18 bases long andusually fewer than 100 bases, and more usually fewer than 60 bases. Theprimer will be chosen to bind substantially uniquely to a targetsequence under the conditions of primer extension, so that the sequencewill normally be one that is conserved or the primer is long enough tobind in the presence of a few mismatches, usually fewer than about 10number % mismatches. By knowing the sequence, which is upstream from thesnp of interest, one may select a sequence, which has a high G-C ratio,so as to have a high binding affinity for the target sequence. Inaddition, the primer should bind reasonably close to the snp, usuallynot more than about 200 bases away, more usually not more than about 100bases away, and preferably within about 50 bases. Since the farther awaythe primer is from the snp, the greater amount of dNTP's, which will beexpended, there will usually be no advantage in having a significantdistance between the primer and the snp detection sequence. Generally,the primer will be at least about 5 bases away from the snp.

[0127] The number of reagent pairs may be varied widely, from a singlepair to two or more pairs, usually at least about 5 pairs, more usuallyat least about 9 pairs and may be 20 pairs or more. By virtue of the useof different e-tags, which have different mobilities and are readilyresolvable under conventional capillary electrophoretic conditions, thesubject pairs may be used to perform multiplexed operations in a singlevessel, where a family of snps may be identified. Usually, the totalnumber of different reagent pairs or different target sequences in asingle determination will be under 200, more usually under 100 and inmany cases will not exceed 50

[0128] In one snp determination protocol, the primer includes thecomplementary base of the snp. This protocol is referred to as “Invader”technology and is described in U.S. Pat. No. 6,001,567. The protocolinvolves providing: (a) (i) a cleavage means, which is normally anenzyme, referred to as a cleavase, that recognizes a triplex consistingof the target sequence, a primer which binds to the target sequence andterminates at the snp position and a labeled probe that bindsimmediately adjacent to the primer and is displaced from the target atthe snp position, when a snp is present; the cleavase clips the labeledprobe at the site of displacement, releasing the label; ii) a source oftarget nucleic acid, the target nucleic acid having a first region, asecond region and a third region, wherein the first region is downstreamfrom the second region and the second region is contiguous to anddownstream from the third region; and (iii) first and secondoligonucleotides having 3′ and 5′ portions, wherein the 3′ portion ofthe first oligonucleotide contains a sequence complementary to the thirdregion of the target nucleic acid and the 5′ portion of the firstoligonucleotide and the 3′ portion of the second oligonucleotide eachcontain sequences usually fully complementary to the second region ofthe target nucleic acid, and the 5′ portion of the secondoligonucleotide contains sequence complementary to the first region ofsaid target nucleic acid; (b) mixing, in any order, the cleavage means,the target nucleic acid, and the first and second oligonucleotides underhybridization conditions that at least the 3′ portion of the firstoligonucleotide is annealed to the target nucleic acid and at least the5′ portion of the second oligonucleotide is annealed to any targetnucleic acid to from a cleavage structure, where the combined meltingtemperature of the complementary regions within the 5′ and 3′ portionsof the first oligonucleotide when annealed to the target nucleic acid isgreater than the melting temperature of the 3′ portion of the firstoligonucleotide and cleavage of the cleavage structure occurs togenerate labeled products; and (c) detecting the labeled cleavageproducts.

[0129] Thus, in an Invader assay attachment of an e-tag to the 5′ end ofthe detector sequence results in the formation of e-tag labelednucleotide when target sequence is present. The e-tag labeled nucleotideis separated and detected. By having a different e-tag for each nucleicacid sequence of interest, having a different electrophoretic mobility,which may require further treatment depending on the total number ofsnp's or target sequences to be detected, one can readily determine thesnp's or measure multiple sequences, which are present in a sample.

[0130] In another snp detection protocol, an alternative method ofcleavage is used and various detectable tags may be employed, the mostcommon using a fluorescent label. The difference in protocol between afluorescent label and another type of label, such as an electrochemicallabel, is the method of detection. Otherwise, the protocols will besubstantially the same. The tagged snp detection sequence will be chosento bind to the target sequence comprising the snp. The length of the snpdetector sequence is in part related to the length and binding affinityof the primer. The two sequences act together to ensure that the pair ofreagents bind to the proper target sequence. The greater the fidelity ofbinding of one member of the pair, the less fidelity that is requiredfor the other member of the pair. Since the observed signal will bedependent upon both members of the pair being present, each memberserves as a check on the other member for production of the signal.However, since except for the cost, it is relatively easy to makereasonably long oligonucleotides, usually both members of the pair willprovide for unique binding to their respective target sequences.Therefore, the length of the snp detector sequence will come within theparameters indicated for the primer, but the total number of bases forthe two pair members will usually be at least 36, more usually at leastabout 40.

[0131] Each snp detection sequence will have at least one nucleotidemodified with an e-tag, which is labeled, which is fluorescent or can besubsequently made fluorescent, or can be detected electrochemically orby other convenient detection methodologies. Usually, the modifiednucleotide will be at the 5′-end of the sequence, but the modifiednucleotide may be anywhere in the sequence, particularly where there isa single nuclease susceptible linkage in the detection sequence. Sincethe determination is based on the at least partial degradation of thesnp detector sequence, having the modified nucleotide at the end ensuresthat if degradation occurs, the e-tag will be released. Since nucleasesmay clip at other than the terminal phosphate link, it is desirable toprevent cleavage at other than the terminal phosphate link. In this wayone avoids the confusion of having the same e-tag joined to differentnumbers of nucleotides after cleavage. Cleavage at the terminalphosphate can be relatively assured by using a linker at the penultimatenucleoside, which is not cleaved by the nuclease, more particularlyhaving only the ultimate linkage susceptible to hydrolysis by anuclease. For example, one may use a thiophosphate, phosphinate,phosphoramidate, or a linker other than a phosphorous acid derivative,such as an amide, boronate, or the like. The particular hydrolaseresistive linker will be primarily one of synthetic convenience, so longas degradation of the binding affinity is not sacrificed. If desired allof the linkers other than the ultimate linker may be resistant tonuclease hydrolysis.

[0132] If desired, the snp detection sequence may have a combination ofa quencher and a fluorescer. In this instance the fluorescer would be inproximity to the nucleoside to which the linker is bonded, as well asthe quencher, so that in the primer extension mixture, fluorescence fromfluorescer bound to the snp detection sequence would be quenched. As thereaction proceeds and fluorescer is released from the snp detectionsequence and, therefore, removed from the quencher, it would then becapable of fluorescence. By monitoring the primer extension mixture forfluorescence, one would be able to determine when there would probablybe a sufficient amount of individual e-tags to provide a detectablesignal for analysis. In this way, one could save time and reagent byterminating the primer extension reaction at the appropriate time. Thereare many quenchers that are not fluorescers, so as to minimizefluorescent background from the snp detection sequence. Alternatively,one could take small aliquots and monitor the reaction for observablee-tags.

[0133] The snp detection sequence may be further modified to improveseparation and detection of the e-tags. By virtue of the difference inmobility of the e-tags, the snp detection sequences will also havedifferent mobilities. Furthermore, these molecules will be present inmuch larger amounts than the released e-tags, so that they may obscuredetection of the released e-tags. Also, it is desirable to havenegatively charged snp detection sequence molecules, since they providefor higher enzymatic activity and decrease capillary wall interaction.Therefore, by providing that the intact snp detection sequence moleculecan be modified with a positively charged moiety, but not the releasede-tag, one can change the electrostatic nature of the snp detectionsequence molecules during the separation. By providing for a ligand onthe snp detection sequence molecule to which a positively chargedmolecule can bind, one need only add the positively charged molecule tochange the electrostatic nature of the snp detection sequence molecule.Conveniently, one will usually have a ligand of under about 1 kDal. Thismay be exemplified by the use of biotin as the ligand and avidin, whichis highly positively charged, as the receptor/positively chargedmolecule. Instead of biotin/avidin, one may have other pairs, where thereceptor, e.g. antibody, is naturally positively charged or is made soby conjugation with one or more positively charged entities, such asarginine, lysine or histidine, ammonium, etc. The presence of thepositively charged moiety has many advantages in substantially removingthe snp detection sequence molecules from the electropherogram. Incarrying out the process, the positively charged moiety is added at orafter the completion of the digestion.

[0134] If desired, the receptor may be used to physically sequester themolecules to which it binds, removing entirely intact e-tags containingthe target-binding region or modified target-binding regions retainingthe ligand. These modified target-binding regions may be as a result ofdegradation of the starting material, contaminants during thepreparation, aberrant cleavage, etc. or other nonspecific degradationproducts of the target binding sequence. As above, a ligand, exemplifiedby biotin, is attached to the target-binding region, e.g. thepenultimate nucleoside, so as to be separated from the e-tag uponcleavage. After the 5′ nuclease assay, a receptor for the ligand, forbiotin exemplified by strept/avidin (hereafter “avidin”) is added to theassay mixture. Other receptors include natural or synthetic receptors,such as immunoglobulins, lectins, enzymes, etc. Desirably, the receptoris positively charged, naturally as in the case of avidin, or is madeso, by the addition of a positively charged moiety or moieties, such asammonium groups, basic amino acids, etc. Avidin binds to the biotinattached to the detection probe and its degradation products. Avidin ispositively charged, while the cleaved e-tag is negatively charged. Thusthe separation of the cleaved e-tag from, not only uncleaved probe, butalso its degradation products, is easily achieved by using conventionalseparation methods. Alternatively, the receptor may be bound to a solidsupport or high molecular weight macromolecule, such as a vessel wall,particles, e.g. magnetic particles, cellulose, agarose, etc., andseparated by physical separation or centrifugation, dialysis, etc. Thismethod further enhances the specificity of the assay and allows for ahigher degree of multiplexing.

[0135] While the ligand may be present at a position other than thepenultimate position and one may make the ultimate linkage nucleaseresistant, so that cleavage is directed to the penultimate linkage, thiswill not be as efficient as having cleavage at the ultimate linkage. Theefficiency would be even worse where the ligand is at a more distantnucleotide from the e-tag. Therefore, while such protocols are feasible,and may be used, they will not be preferred.

[0136] As a general matter, one may have two ligands, if the nature ofthe target-binding region permits. As described above, one ligand can beused for sequestering e-tags bound to target-binding region retainingthe first ligand from products lacking the first ligand. Isolation andconcentration of the e-tags bound to a modified target-binding regionlacking the first ligand would then be performed. In using the twoligands, one would first combine the reaction mixture with a firstreceptor for the first ligand for removing target-binding regionretaining the first ligand. One could either separate the first receptorfrom the composition or the first receptor would be retained in thecomposition, as described. This would be followed by combining theresulting composition, where the target-binding region containing thefirst ligand is bound to the first receptor, with the second receptor,which would serve to isolate or enrich for modified target-bindingregion lacking the first ligand, but retaining the second ligand. Thesecond ligand could be the detectable label; a small molecule for whicha receptor is available, e.g. a hapten, or a portion of the e-tag couldserve as the second ligand. After the product is isolated or enriched,the e-tag could be released by denaturation of the receptor,displacement of the product, high salt concentrations and/or organicsolvents, etc.

[0137] For e-tags associated with nucleic acids sequences, improvementsinclude employing a blocking linkage between nucleotides in thesequence, particularly at least one of the links between the second tofourth nucleotides to inhibit cleavage at this or subsequent sites, andusing control sequences for quantitation. Further improvements in thee-tags provide for having a positively multicharged moiety joined to thee-tag probe during separation.

[0138] The above three methods are generally applicable not only togenerating a single e-tag per sequence detected but also to generationof a single oligonucleotide fragment for fragment separation andidentification by electrophoresis or by mass spectra as it is essentialto get one fragment per sequence detected. For purpose of explanation,these methods are illustrated below.

[0139] The complementary base to the snp may be anywhere in the detectorsequence, desirably at other than the terminal nucleoside to enhance thefidelity of binding. The snp detector sequence will be designed toinclude adjacent nucleotides, which provide the desired affinity for thehybridization conditions. The snp detection sequence may be synthesizedby any convenient means, such as described in Matthews, et al., AnalBiochem. (1988) 169:1-25; Keller, et al., “DNA Probes,” 2^(nd) edition(1993) Stockton Press, New York, N.Y.; and Wetmur, Critical Reviews inBiochemistry and Molecular Biology (1991) 26:227-259.

[0140] The extension reaction is performed by bringing together thenecessary combination of reagents and subjecting the mixture toconditions for carrying out the desired primer extension. Suchconditions depend on the nature of the extension, e.g., PCR, singleprimer amplification, LCR, NASBA, 3SR and so forth, where the enzymewhich is used for the extension has 5′-3′ nuclease activity. Theextension reaction may be carried out as to both strands or as to only asingle strand. Where pairs of primer and snp detection sequence are usedfor both strands, conveniently, the e-tag will be the same, but thebases will be different. In this situation, one may wish to have acleavable linkage to the base, so that for the same snp, one wouldobtain the same e-tag. Alternatively, if the number of snps to bedetermined is not too high, one could use different c-tags for each ofthe strands. Usually, the reaction will be carried out by usingamplifying conditions, so as to provide an amplified signal for eachsnp. Amplification conditions normally employ thermal cycling, whereafter the primer extension and release of e-tags associated with snpswhich are present, the mixture is heated to denature the double-strandedDNA, cooled, where the primer and snp detection sequence can rehybridizeand the extension repeated.

[0141] Depending on the protocol, the e-tags or e-tag, will be separatedfrom a portion or substantially all of the detection sequence, usuallyretaining not more than about 3 nucleotides, more usually not more thanabout 2 nucleotides and preferably from 0 to 1 nucleotide. By having acleavable linker between the e-tag and the detection sequence, the e-tagmay be freed of all the nucleotides. By having a nuclease resistantpenultimate link, a single nucleotide may be bonded to the e-tag.

[0142] Reagents for conducting the primer extension are substantiallythe same reaction materials for carrying out an amplification, such asan amplification indicated above. The nature and amounts of thesereagents are dependent on the type of amplification conducted. Inaddition to oligonucleotide primers the reagents also comprisenucleoside triphosphates and a nucleotide polymerase having 5′-3′nuclease activity.

[0143] The nucleoside triphosphates employed as reagents in anamplification reaction include deoxyribonucleoside triphosphates such asthe four common deoxyribonucleoside triphosphates dATP, dCTP, dGTP anddTTP. The term “nucleoside triphosphates” also includes derivatives andanalogs thereof, which are exemplified by those derivatives that arerecognized and polymerized in a similar manner to the underivatizednucleoside triphosphates.

[0144] The nucleotide polymerase employed is a catalyst, usually anenzyme, for forming an extension of an oligonucleotide primer along apolynucleotide such as a DNA template, where the extension iscomplementary thereto. The nucleotide polymerase is a template dependentpolynucleotide polymerase and utilizes nucleoside triphosphates asbuilding blocks for extending the 3′-end of a polynucleotide to providea sequence complementary with the polynucleotide template. Usually, thecatalysts are enzymes, such as DNA polymerases, for example, prokaryoticDNA polymerase (I, II, or III), T4 DNA polymerase, T7 DNA polymerase,Vent DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and thelike. Polymerase enzymes may be derived from any source, such aseukaryotic or prokaryotic cells, bacteria such as E. coli, plants,animals, virus, thermophilic bacteria, genetically modified enzymes, andso forth.

[0145] The conditions for the various amplification procedures are wellknown to those skilled in the art. In a number of amplificationprocedures, thermal cycling conditions as discussed above are employedto amplify the polynucleotides. The combination of reagents is subjectedto conditions under which the oligonucleotide primer hybridizes to thepriming sequence of, and is extended along, the correspondingpolynucleotide. The exact temperatures can be varied depending on thesalt concentration, pH, solvents used, length of and composition of thetarget polynucleotide sequence and the oligonucleotide primers.

[0146] Thermal cycling conditions are employed for conducting anamplification involving temperature or thermal cycling and primerextension, such as in PCR or single primer amplification, and the like.The pH and the temperature are selected so as to cause, eithersimultaneously or sequentially, dissociation of any internallyhybridized sequences, hybridization or annealing of the oligonucleotideprimer and the snp detection sequence with the target polynucleotidesequence, extension of the primer, release of the e-tag from snpdetection sequence bound to the target polynucleotide sequence anddissociation of the extended primer. This usually involves cycling thereaction medium between two or more temperatures. In conducting such amethod, the medium is cycled between two to three temperatures. Thetemperatures for thermal cycling generally range from about 50° C. to100° C., more usually, from about 60° C. to 95° C. Relatively lowtemperatures of from about 30° C. to about 65° C. can be employed forthe extension steps, while denaturation and hybridization can be carriedout at a temperature of from about 50° C. to about 105° C. The reactionmedium is initially at about 20° C. to about 45° C., preferably, about25° C. to about 35° C. Relatively low temperatures of from about 50° C.to about 80° C., preferably, 50° C. to about 60° C., are employed forthe hybridization or annealing steps, while denaturation is carried outat a temperature of from about 80° C. to about 100° C., preferably, 90°C. to about 95° C., and extension is carried out at a temperature offrom about 70° C. to about 80° C., usually about 72° C. to about 74° C.The duration of each cycle may vary and is usually about 1 to 120seconds, preferably, about 5 to 60 seconds for the denaturation steps,and usually about 1 to 15 seconds, preferably, about 1 to 5 seconds, forthe extension steps. It is to be understood that the actual temperatureand duration of the cycles employed are dependent on the particularamplification conducted and are well within the knowledge of thoseskilled in the art.

[0147] Generally, an aqueous medium is employed. Other polar cosolventsmay also be employed, usually oxygenated organic solvents of from 1-6,more usually from 1-4, carbon atoms, including alcohols, ethers,formamide and the like. Usually, these cosolvents, if used, are presentin less than about 70 weight percent, more usually in less than about 30weight percent.

[0148] The pH for the medium is usually in the range of about 4.5 to9.5, more usually in the range of about 5.5 to 8.5, and preferably inthe range of about 6 to 8. Various buffers may be used to achieve thedesired pH and maintain the pH during the determination. Illustrativebuffers include borate, phosphate, carbonate, Tris, barbital and thelike. The particular buffer employed is not critical to this inventionbut in individual methods one buffer may be preferred over another. Themedium may also contain materials required for enzyme activity such as adivalent metal ion (usually magnesium).

[0149] The selection of the snp detection sequence will affect thestringency employed during the primer extension, particularly at thestage of hybridization. Since in a substantial number of samples, theDNA will be heterozygous for snps, rather than homozygous, one does notwish to have false positives, where the snp detection sequence may bondto the sequence comprising the prevalent nucleotide, as well as thesequence comprising the snp. Where the DNA sample is homozygous for theprevalent sequence, it is also important that the snp detection sequencedoes not bind to give a false positive. Therefore, the difference inT_(m) between the snp containing sequence and the wild-type sequencewill usually be at least about 3° C., more usually at least about 5° C.,under the conditions of the primer extension.

[0150] Various ancillary materials will frequently be employed in themethods in accordance with the present invention. For example, inaddition to buffers and salts, the medium may also comprise stabilizersfor the medium and the reaction components. Frequently, the medium mayalso include proteins such as albumins, quaternary ammonium salts,polycations such as spermine, surfactants, particularly non-ionicsurfactants, binding enhancers, e.g., polyalkylene glycols, or the like.

[0151] The reaction is conducted for a time sufficient to produce thedesired number of copies of each of the polynucleotides suspected ofbeing present as discussed below. Generally, the time period forconducting the entire method will be from about 10 to 200 minutes. Asmentioned above, it is usually desirable to minimize the time period.

[0152] The concentration of the nucleotide polymerase is usuallydetermined empirically. Preferably, a concentration is used that issufficient such that the amplification is robust. The primary limitingfactor generally is the cost of the reagent. Such enzymes include PfuDNA polymerase (native and recombinant) from Stratagene, La Jolla,Calif., Ultma DNA polymerase from Perkin Elmer, Foster City, Calif.,rBst DNA polymerase from Epicentre Technologies, Madison, Wis., VENT DNApolymerase from New England Biolabs, Beverly, Mass., Tli DNA polymerasefrom Promega Corp., Madison, Wis., and Pwo DNA polymerase fromBoehringer Mannheim, Indianapolis; IN, and the like.

[0153] The initial concentration of each of the polynucleotidescontaining the respective target snps can be as low as about 50 pg/ml ina sample. After amplification the concentration of each polynucleotideshould be at least about 10 picomolar, generally in the range of about10 pM to about 10 nM, usually from about 10 to 1010, more usually fromabout 10³ to 10⁸ molecules in a sample preferably at least 10⁻²¹M in thesample and may be 10⁻¹⁰ to 10⁻¹⁹M, more usually 10⁻¹⁴ to 10⁻¹⁹M. Ingeneral, the reagents for the reaction are provided in amounts toachieve extension of the oligonucleotide primers.

[0154] The concentration of the oligonucleotide primer(s) will be about1 to about 20 μM and is usually about 1 to about 10 μM, preferably,about 1 to about 4 μM, for a sample size that is about 10 fM.Preferably, the concentration of the oligonucleotide primer(s) issubstantially in excess over, preferably at least about 10⁷ to about10¹⁰ times greater than, more preferably, at least about 10⁹ timesgreater than, the concentration of the corresponding targetpolynucleotides.

[0155] The amount of the oligonucleotide probes will be 10 to about 500nM and is usually about 50 to about 200 nM for a sample size that isabout 10 fM (10 fg/μl). Preferably, the concentration of theoligonucleotide probes is substantially in excess over, preferably atleast about 10⁷ times greater than, more preferably, at least about 10⁸times greater than, the concentration of each of the targetpolynucleotides.

[0156] The concentration of the nucleoside triphosphates in the mediumcan vary widely; preferably, these reagents are present in an excessamount. The nucleoside triphosphates are usually present in about 100 μMto about 1 mM, preferably, about 20 to about 400 μM

[0157] The order of combining of the various reagents to form thecombination may vary. Usually, the sample containing the polynucleotidesis combined with a pre-prepared combination of nucleoside triphosphatesand nucleotide polymerase. The oligonucleotide primers and the snpdetection sequences may be included in the prepared combination or maybe added subsequently. However, simultaneous addition of all of theabove, as well as other step-wise or sequential orders of addition, maybe employed provided that all of the reagents described above arecombined prior to the start of the reactions. The oligonucleotide pairsmay be added to the combination of the reagents at or prior to theinitiation of the primer extension reaction and may be replenished fromtine-to-time during the primer extension reaction.

[0158] For quantitation, one may choose to use controls, which provide asignal in relation to the amount of the target that is present or isintroduced. Where one is dealing with a mixture of nucleic acidmolecules, as in the case of mRNA in a lysate, one may use the knownamounts of one or more different mRNAs in the particular cell types asthe standards. Desirably, one would have at least two controls,preferably at least 3 controls, where the variation in number betweenany two controls is at least about 10², and the total range is at leastabout 10³, usually at least about 10⁴. However, determining theconsistent ratio of mRNAs occurring naturally will result in a largemargin of error, so that one would usually rely on synthetic targets.Where a control system is added for quantitation, as compared to relyingon the presence of a known amount of a plurality of endogenous nucleicacids, the control system will comprise at least two control sequences,usually at least 3 control sequences and generally not more than about 6control sequences, where the upper limit is primarily one of convenienceand economy, since additional control sequences will usually not addsignificant additional precision which will usually be at least about 50nucleotides, more usually at least about 100 nucleotides. The controlsequences will have a common primer sequence and different controldetection sequences, which are intended to parallel the primer sequenceand snp detection sequence in size, spacing and response to the primerextension conditions. In carrying out the primer extension reaction withsample nucleic acid, one would then add different number of molecules ofthe different control sequences, so that one could graph the result togive a signal/number relationship. This graph could then be used torelate signals observed with target molecules to the number of moleculespresent.

[0159] After completion of the primer extension reaction, either bymonitoring the change in fluorescence as described above or takingaliquots and assaying for total free e-tags, the mixture may now beanalyzed. Depending on the instrument, today from one to four differentfluorescers activated by the same light source and emitting at differentdetectable labels may be used. With improvements, five or more differentfluorescers will be available, where an additional light source may berequired. Electrochemical detection is described in U.S. Pat. No.6,045,676.

[0160] The subject assays are predicated on having a reagent that has ahigh affinity for a reciprocal binding member, the analyte. Usually, thebinding affinity will be at least about 10⁻⁷M⁻¹, more usually, at leastabout 10⁻⁸M⁻¹. For the most part, the reagents will be receptors, whichincludes antibodies, IgA, IgD, IgG, IgE and IgM and subtypes thereof,enzymes, lectins, nucleic acids, nucleic acid binding proteins, or anyother molecule that provides the desired specificity for the analyte inthe assay, one of the members normally being a protein. The antibodiesmay be polyclonal or monoclonal or mixtures of monoclonal antibodiesdepending on the nature of the target composition and the targets. Thetargets or analytes may be any molecule, such as small organic moleculesof from about 100 to 2500 Dal, poly(amino acids) including peptides offrom about 3 to 100 amino acids and proteins of from about 100 to 50,000or more amino acids, saccharides, lipids, nucleic acids, etc., where theanalytes may be part of a larger assemblage, such as a cell, microsome,organelle, virus, protein complex, chromosome or fragment thereof,nucleosome, etc.

[0161] In addition, the subject heterogeneous assays require that theunbound labeled reagent be separable from the bound labeled reagent.This can be achieved in a variety of ways. Each way requires that areagent that distinguishes between the complex of labeled reagent andtarget be bound to a solid support. The solid support may be a vesselwall, e.g. microtiter well plate well, capillary, plate, slide, beads,including magnetic beads, liposomes, or the like. The primarycharacteristics of the solid support is that it permits segregation ofthe bound labeled specific binding member from unbound and does notinterfere with the formation of the binding complex, nor the otheroperations of the determination.

[0162] The solid support may have the complex directly bound to thesupport or indirectly bound. For directly bound, one may have thebinding member covalently or non-covalently bound to the support. Forproteins, many surfaces provide non-diffusible binding of a protein tothe support, so that one adds the protein to the support and allows theprotein to bind, washes away weakly bound protein and then adds aninnocuous protein to coat any actively binding areas that are stillavailable. The surface may be activated with various functionalitiesthat will form covalent bonds with a binding member. These groups mayinclude imino halides, activated carboxyl groups, e.g. mixed anhydridesor acyl halides, amino groups, α-halo or pseudohaloketones, etc. Thespecific binding member bound to the surface of the support may be anymolecule which permits the binding portion of the molecule, e.g.epitope, to be available for binding by the reciprocal member. Where thebinding member is polyepitopic, e.g. proteins, this is usually less of aproblem, since the protein will be polyepitopic and even with randombinding of the protein to the surface, the desired epitope will beavailable for most of the bound molecules. For smaller molecules,particularly under 5 kDal, one will usually have an active functionalityon the specific binding member that preserves the binding site, wherethe active functionality reacts with a functionality on the surface ofthe support. The same functionalities described above may find use.Conveniently, one may use the same site for preparing the conjugateimmunogen to produce antibodies, as the site for the activefunctionality for linking to the surface.

[0163] The assays may be performed in a competitive mode or a sandwichmode. In the competitive mode, one has the target competing with alabeled binding member for the reciprocal member, which reciprocalmember is bound to the support, either during the complex formation orafter, e.g. where antibody is a specific binding member and anti(Ig_(H))is bound to the support. In this mode, the binding sites of thereciprocal binding member become at least partially filled by thetarget, reducing the number of available binding sites for the labeledreciprocal binding member. Thus, the number of labeled binding membersthat bind to the reciprocal binding member will be in direct proportionto the number of target molecules present. In the sandwich mode, thetarget is able to bind at the same time to different binding members; afirst support bound member and a second member which binds at a site ofthe target molecule different from the site at which the support boundmember binds. The resulting complex has three components, where thetarget serves to link the labeled binding member to the support.

[0164] In carrying out the assays, the components are combined, usuallywith the target composition added first and then the labeled members inthe competitive mode and in any order in the sandwich mode. Usually, thelabeled member in the competitive mode will be equal to at least 50% ofthe highest number of target molecules anticipated, preferably at leastequal and may be in 2-10 fold excess or greater. The particular ratio oftarget molecules to labeled molecules will depend on the bindingaffinities, the length of time the mixture is incubated, the off ratesfor the target molecule with its reciprocal binding member, the size ofthe sample and the like. In the case of the sandwich assays, one willhave at least an equal amount of the labeled binding member to thehighest expected amount of the target molecules, usually at least 1.5fold excess, more usually at least 2 fold excess and may have 10 foldexcess or more. The components are combined under binding conditions,usually in an aqueous medium, generally at a pH in the range of 5-10,with buffer at a concentration in the range of about 10 to 200 mM. Theseconditions are conventional, where conventional buffers may be used,such as phosphate, carbonate, HEPES, MOPS, Tris, borate, etc., as wellas other conventional additives, such as salts, stabilizers, organicsolvents, etc.

[0165] Usually, the unbound labeled binding member will be removed bywashing the bound labeled binding member. Where particles or beads areemployed, these may be separated from the supernatant before washing, byfiltration, centrifugation, magnetic separation, etc. After washing, thesupport may be combined with a liquid into which the e-tags are to bereleased and/or the functionality of the e-tags is reacted with thedetectable label, followed by or preceded by release. Depending on thenature of the cleavable bond and the method of cleavage, the liquid mayinclude reagents for the cleavage. Where reagents for cleavage are notrequired, the liquid is conveniently an electrophoretic buffer. Forexample, where the cleavable linkage is photo labile, the support may beirradiated with light of appropriate wavelength to release the e-tags.Where detectable labels are not present on the e-tags, the e-tags may bereacted with the detectable labels. In some instances the detectablelabel may be part of the reagent cleaving the cleavable bond, e.g. adisulfide with a thiol. Where there is a plurality of differentfunctionalities on different binding members for reaction with thelabel, the different labels will have functionalities that react withone of the functionalities. The different labels may be added togetheror individually in a sequential manner. For example, where thefunctionalities involve thiols, carboxyl groups, aldehydes and olefins,the labels could have activated olefins, alcohols, amines and thiolgroups, respectively. By having removable protective groups for one ormore of the functionalities, the protective groups may be removedstepwise and the labels added stepwise. In this way cross-reactivity maybe avoided. Whether one has the detectable label present initially orone adds the detectable label is not critical to this invention and willfrequently be governed by the nature of the target composition, thenature of the labeled binding members, and the nature of the detectablelabels. For the most part, it will be a matter of convenience as to theparticular method one chooses for providing the detectable labelede-tag.

[0166] Where a reagent is necessary for cleavage, the e-tags may berequired to be separated from the reagent solution, where the reagentinterferes with the electrophoretic analysis. Depending on the nature ofthe e-tags and the reagent, one may sequester the e-tags from thereagent by using ion exchange columns, liquid chromatography, an initialelectrophoretic separation, and the like. Alternatively, as discussedpreviously, one may have a ligand bound to the e-tag or retained portionof the target-binding region for isolating the e-tag, so as to removeany interferents in the mixture. Once the solution of e-tags is preparedand free of any interfering components, the solution may be analyzedelectrophoretically. The analysis may employ capillary electrophoresisdevices, microfluidic devices or other devices that can separate aplurality of compounds electrophoretically, providing resolved bands ofthe individual e-tags.

[0167] The protocols for the subject homogeneous assays will follow theprocedures for the analogous assays, which may or may not include areleasable tag. These protocols employ a signal producing system thatincludes the label on one of the binding members, the cleavable bondassociated with the e-tag, electromagnetic radiation or other reagentsinvolved in the reaction or for diminishing background signal. In assaysinvolving the production of hydrogen peroxide, one may wish to have amolecule in solution that degrades hydrogen peroxide to prevent reactionbetween hydrogen peroxide produced by a label bound to an analytemolecule and an e-tag labeled binding member that is not bound to thesame analyte molecule.

[0168] Generally, the concentrations of the various agents involved withthe signal producing system will vary with the concentration range ofthe individual analytes in the samples to be analyzed, generally beingin the range of about 10 nM to 10 mM. Buffers will ordinarily beemployed at a concentration in the range of about 10 to 200 mM. Theconcentration of each analyte will generally be in the range of about 1pM to about 100 μM, more usually in the range of about 100 pM to 10 μM.Although in specific situations the concentrations may be higher orlower, depending on the nature of the analyte, the affinity of thereciprocal binding members, the efficiency of release of the e-tags, thesensitivity with which the e-tags are detected, and the number ofanalytes, as well as other considerations.

[0169] The reactive species that is produced in the assay, analogous tothe subject assay, is employed in a different way than was used in theanalogous assay, but otherwise the conditions will be comparable. Inmany instances, the chemiluminescent compound when activated will resultin cleavage of a bond, so that one may obtain release of the e-tag.Assays that find use are described in U.S. Pat. Nos. 4,233,402;5,616,719; 5,807,675; and 6,002,000. One would combine the analyte withone or both reagents. The particular order of addition will vary withthe nature of the reagents. Generally, one would prefer to combine thebinding reagents and the sample and allow the mixture to incubate,generally at least about 5 min, more usually at least about 15 min,before irradiating the mixture or adding the remaining reagents.

[0170] One may also use the subject libraries to analyze the effect ofan agent on a plurality of different compounds. For example, one mayprepare a plurality of substrates labeled with an e-tag, where theenzyme catalyzes a reaction resulting in a change in mobility betweenthe product and the starting material. These assays can find use indetermining affinity groups or preferred substrates for hydrolases,oxidoreductases, lyases, etc. For example, with kinases andphosphatases, one adds or removes a charged group, so as to change themobility of the product. By preparing a plurality of alcohols orphosphate esters, one can determine which of the compounds serves as asubstrate. By labeling the substrates with e-tags, one can observe theshift from the substrate to the product as evidence of the activity of acandidate substrate with the enzyme. By preparing compounds as suicideinhibitors, the enzymes may be sequestered and the e-tags released todefine those compounds that may serve as suicide inhibitors and,therefore, preferentially bind to the active site of the enzyme.

[0171] One may also use the subject methods for screening for theactivity of one or more candidate compounds, particularly drugs, fortheir activity against a battery of enzymes. In this situation, onewould use active substrates for each of the enzymes to be evaluated,where each of the substrates would have its own e-tag. For those enzymesfor which the drug is an inhibitor, the amount of product would bediminished in relation to the amount of product in the absence of thecandidate compound. In each case the product would have a differentmobility from the substrate, so that the substrates and products couldbe readily distinguished by electrophoresis. By appropriate choice ofsubstrates and detectable labels, one would obtain electropherogramsshowing the effect of the candidate compound on the activity of thedifferent enzymes.

[0172] In those instances where a fluorescent label is not present onthe e-tag bound to the product comprising the mir, the mixture may beadded to functionalized fluorescent tags to label the e-tag with afluorescer. For example, where a thiol group is present, the fluorescercould have an activated ethylene, such as maleic acid to form thethioether. For hydroxyl groups, one could use activated halogen orpseudohalogen for forming an ether, such as an α-haloketone. Forcarboxyl groups, carbodiimide and appropriate amines or alcohols wouldform amides and esters, respectively. For an amine, one could useactivated carboxylic acids, aldehydes under reducing condtions,activated halogen or pseudohalogen, etc. When synthesizingoligopeptides, protective groups are used. These could be retained whilethe fluorescent moiety is attached to an available functionality on theoligopeptide.

[0173] The presence of each of the released or intact e-tags isdetermined by the label. The separation of the mixture of labeled e-tagsis carried out by electroseparation, which involves the separation ofcomponents in a liquid by application of an electric field, preferably,by electrokinesis (electrokinetic flow), electrophoretic flow,electroosmotic flow or combination thereof, with the separation of thee-tag mixture into individual fractions or bands. Electroseparationinvolving the migration and separation of molecules in an electric fieldis based on differences in mobility. Various forms of electroseparationinclude, by way of example and not limitation, free zoneelectrophoresis, gel electrophoresis, isoelectric focusing andisotachophoresis. Capillary electroseparation involveselectroseparation, preferably by electrokinetic flow, includingelectrophoretic, dielectrophoretic and/or electroosmotic flow, conductedin a tube or channel of about 1-200 μm, usually, about 10-100 μmcross-sectional dimensions. The capillary may be a long independentcapillary tube or a channel in a wafer or film comprised of silicon,quartz, glass or plastic.

[0174] In capillary electroseparation, an aliquot of the reactionmixture containing the e-tag products is subjected to electroseparationby introducing the mixture or an aliquot into an electroseparationchannel that may be part of, or linked to, a capillary device in whichthe amplification and other reactions are performed. An electricpotential is then applied to the electrically conductive mediumcontained within the channel to effectuate migration of the componentswithin the combination. Generally, the electric potential applied issufficient to achieve electroseparation of the desired componentsaccording to practices well known in the art. One skilled in the artwill be capable of determining the suitable electric potentials for agiven set of reagents used in the present invention and/or the nature ofthe cleaved labels, the nature of the reaction medium and so forth. Theparameters for the electroseparation including those for the medium andthe electric potential are usually optimized to achieve maximumseparation of the desired components. This may be achieved empiricallyand is well within the purview of the skilled artisan.

[0175] Capillary devices are known for carrying out amplificationreactions such as PCR. See, for example, Analytical Chemistry (1996)68:4081-4086. Devices are also known that provide functional integrationof PCR amplification and capillary electrophoresis in a microfabricatedDNA analysis device. One such device is described by Woolley, et al., inAnal. Chem. (1996) 68:4081-4086. The device provides a microfabricatedsilicon PCR reactor and glass capillary electrophoresis chips. In thedevice a PCR chamber and a capillary electrophoresis chip are directlylinked through a photolithographically fabricated channel filled with asieving matrix such as hydroxyethylcellulose. Electrophoretic injectiondirectly from the PCR chamber through the cross injection channel isused as an “electrophoretic valve” to couple the PCR and capillaryelectrophoresis devices on a chip.

[0176] The capillary electrophoresis chip contains a sufficient numberof main or secondary electrophoretic channels to receive the desirednumber of aliquots from the PCR reaction medium or the solutionscontaining the e-tags, etc., at the intervals chosen.

[0177] For capillary electrophoresis one may employ one or moredetection zones to detect the separated e-tags. It is, of course, withinthe purview of the present invention to utilize several detection zonesdepending on the nature of the amplification process, the number ofcycles for which a measurement is to be made and so forth. There may beany number of detection zones associated with a single channel or withmultiple channels. Suitable detectors for use in the detection zonesinclude, by way of example, photomultiplier tubes, photodiodes,photodiode arrays, avalanche photodiodes, linear and array chargecoupled device (CCD) chips, CCD camera modules, spectrofluorometers, andthe like. Excitation sources include, for example, filtered lamps,LED's, laser diodes, gas, liquid and solid state lasers, and so forth.The detection may be laser scanned excitation, CCD camera detection,coaxial fiber optics, confocal back or forward fluorescence detection insingle or array configurations, and the like.

[0178] Detection may be by any of the known methods associated with theanalysis of capillary electrophoresis columns including the methodsshown in U.S. Pat. No. 5,560,811 (column 11, lines 19-30), U.S. Pat.Nos. 4,675,300, 4,274,240 and 5,324,401, the relevant disclosures ofwhich are incorporated herein by reference.

[0179] Those skilled in the electrophoresis arts will recognize a widerange of electric potentials or field strengths may be used, forexample, fields of 10 to 1000 V/cm are used with 200-600 V/cm being moretypical. The upper voltage limit for commercial systems is 30 kV, with acapillary length of 40-60 cm, giving a maximum field of about 600 V/cm.For DNA, typically the capillary is coated to reduce electroosmoticflow, and the injection end of the capillary is maintained at a negativepotential, which may be reversed, as appropriate.

[0180] For ease of detection, the entire apparatus may be fabricatedfrom a plastic material that is optically transparent, which generallyallows light of wavelengths ranging from 180 to 1500 nm, usually 220 to800 nm, more usually 450 to 700 nm, to have low transmission losses.Suitable materials include fused silica, plastics, quartz, glass, and soforth.

[0181] In mass spectrometry, the e-tags may be different from the c-tagsused in electrophoresis, since the e-tags do not require a label, nor acharge. Thus, these c-tags may be differentiated solely by mass, whichcan be a result of atoms of different elements, isotopes of suchelements, and numbers of such atoms. In the subject invention, such useof e-tags will be coupled with a process for removing the iterativeextensions of the nucleic acid sequence, where degradation or cleavagehas occurred at a site other than the ultimate linkage.

[0182] One embodiment of a system according to the present invention ispresented in FIG. 10. This figure illustrates a system (100) for thesimultaneous, multiplexed determination of a plurality of events. Eachevent is distinguished from the others by electrophoresis. For example,a snp locus may be characterized using a pair of reagents, each specificfor one allele of the locus. Each reagent is bonded to an e-tag with aunique electrophoretic mobility and an associated label. When thereagent is combined with a sample of interest in a reaction vessel(101), the associated e-tag is modified in a manner that changes itselectrophoretic mobility if its specific target is present. After thereaction, the mixture is moved (102) onto an electrophoretic device(103) for separation of the e-tags contained in the mixture. A powercontrol box (104) is used in conjunction with the device to controlinjection of the sample into the separation channel (105). Each e-tagspecies migrates down the separation channel of the device with amobility unique to that tag, moving past a detector (106) that monitorsits presence by its associated label. The data collected by the detectoris sent to a data processor (107), which determines the presence of eachsnp allele in the sample based on the mobility of its correspondinge-tag.

[0183] In another example, a group of snp loci may be monitored in amultiplexed reaction. In this case, a plurality of pairs of e-tagreagents corresponding to the snp loci are combined with the sample in asingle reaction vessel under conditions where the e-tag is released fromat least a portion of the oligonucleotides sequence to which it isbonded when a pair is bonded to its target. The e-tags are eitherlabeled for detection or the label is added by means of a reactivefunctionality present on the e-tag. The labeled e-tag products of thereaction are resolved from one another on the electrophoretic device,and again are monitored as they move past the detector. The level ofmultiplexing possible in this system is limited only by the degree ofresolution that can be obtained between a designated set of e-tags onthe electrophoretic device.

[0184] An additional degree of flexibility can be conferred on the assayby the stage at which the e-tags are labeled. As described above, eache-tag may already contain a detectable label when introduced to thereaction. Alternatively, an e-tag may contain a functionality allowingit to bind to a label after reaction with the sample is complete (108).In this embodiment, an e-tag comprising a functionality for binding to adetectable label is combined with a sample (101). After a reaction tomodify the mobility of the e-tag if its target is present in the sample,additional reagents are combined in a sample vessel (109) with theproducts of the first reaction, which will react with the modifiede-tag(s) to add a detectable label.

EXAMPLES

[0185] The invention is demonstrated further by the followingillustrative examples. Parts and percentages are by weight unlessotherwise indicated. Temperatures are in degrees Centigrade (° C.)unless otherwise specified. The following preparations and examplesillustrate the invention but are not intended to limit its scope. Unlessotherwise indicated, oligonucleotides and peptides used in the followingexamples were prepared by synthesis using an automated synthesizer andwere purified by gel electrophoresis or HPLC.

[0186] The following abbreviations have the meanings set forth below:

[0187] Tris HC1—Tris(hydroxymethyl)aminomethane-HCl (a 10× solution)from BioWhittaker, Walkersville, Md.

[0188] HPLC—high performance liquid chromatography

[0189] BSA—bovine serum albumin from Sigma Chemical Company, St. LouisMo.

[0190] EDTA—ethylenediaminetetetraacetate from Sigma Chemical Company

[0191] bp—base pairs

[0192] g—grams

[0193] mM—millimolar

[0194] TET—tetrachlorofluorescein

[0195] FAM—fluorescein

[0196] TAMRA—tetramethyl rhodamine

[0197] Reagents:

[0198] TET and FAMRA were purchased from Perkin Elmer (Foster City,Calif.) as were conjugates of TET, FAM and TAMRA with oligonucleotides.

[0199] Master Mix (2×): 20 mM Tris-HCl, 2.0 mM EDTA, pH 8.0 (8%Glycerol), 10 mM MgCl₂, dATP 400 μM, dCTP 400 μM, dGTP 400 μM, dUTP 400μM, AmpliTaq Gold®0.1 U/μl (from Perkin Elmer), Amperase UNG® 0.02 U/μl(from Perkin Elmer)

[0200] Probes and Primers: (10×) Forward Primer: 3.5 μM 5′-TCA CCA CATCCC AGT G-3′ (SEQ ID NO:1) Reverse Primer 2.0 μM 5′-GAG GGA GGTTTGGCTG-3′ (SEQ ID NO:2) Plasmid Allele 1 2.0 μM 5′ TET-CCA GCA ACC AAT GATGCC CGT T-TAMRA-3′ (SEQ ID NO:3) Probe: (200 nM per reaction) PlasmidAllele 2 2.0 μM 5′ FAM-CCA GCA AGC ACT GAT GCC TGT T-TAMRA-3′ (SEQ IDNO:4) Probe: (200 nM per reaction)

[0201] Target DNA:

[0202] Plasmid Allele-1: 10 fg/μl=approximately 1000 copies/μl

[0203] Plasmid Allele-2: 10 fg/μl=approximately 1000 copies/μl

Example 1 The Experiment was Set Up to Run in the Following Fashion (6Samples, a Triplicate for Allele 1 and Another Triplicate for Allele-2)

[0204] 22 μl of Mastermix

[0205] 13 μl of probes and primers (both the probes are present)

[0206] 4.0 μl of Allele-1 or Allele-2

[0207] 11 μl of buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)

[0208] The above volumes were added to a PCR tubes and the reactionmixtures were cycled in the following fashion for 40 cycles.

[0209] Initial Steps:

[0210] The Reaction Mixtures Were Kept at 50° C. for 2 Minutes forOptimal AmpErase UNG Activity. The 10 Minute, 95° C. Step was Requiredto Activate AmpliTaq Gold DNA Polymerase.

[0211] Each of the 40 cycles was performed on a Gene Amp ® system 9600thermal cycler (Perkin Elmer) in the following fashion: MeltAnneal/Extend/Cleave 15 seconds 60 seconds 95° C. 60° C.

[0212] Results from experiments with Allele-1 are shown in FIG. 2. CEseparation of the reaction products of Allele 1 after 0 and 40 cycles.CE instrument was Beckman P/ACE 5000 with LIF detection. BGE: 2.5%LDD30, 7 M urea, 1×TBE. Capillary: 100 μm i.d., 375 μm o.d., Lc=27 cm,Ld=6.9 cm. Detection: λex=488 nm, λem=520 nm. Injection: 5 s at 2.0 kV.Field strength: 100 V/cm at room temperature. Peaks: P=unreacted primer,P′=primer product.

[0213] Results from experiments with Allele-2 are shown in FIG. 3. CEseparation of the reaction products of Allele 2 after 0 and 40 cycles.Experimental conditions were as given above for FIG. 2 experiment exceptfor BGE composition: 2.0% LDD30, 1×TBE.

Example 2 A Multiplexed Reaction with Both Allele 1 and Allele 2 Presentin Equal Ratio

[0214] The experiment was set up in the following fashion (3 reactiontubes, a triplicate)

[0215] 22 μl of Mastermix

[0216] 13 μl of probes and primers (both of the probes were present)

[0217] 4.0 μl of Allele-1

[0218] 4.0 μl of Allele-2

[0219] 7 μl of buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)

[0220] The above volumes were added to a PCR tubes and the reactionmixtures were cycled in the following fashion for 40 cycles.

[0221] Initial Steps:

[0222] The reaction mixtures are kept at 50° C. for 2 minutes foroptimal AmpErase UNG activity. The 10 minute, 95° C. step is required toactivate AmpliTaq Gold DNA Polymerase.

[0223] Each of the 40 cycles is performed in the following fashion MeltAnneal/Extend/Cleave 15 seconds 60 seconds 95° C. 60° C.

[0224] The results are shown in FIG. 4. CE separation of a 1:1 mixtureof the 40 cycles products of Alleles 1 and 2. Experimental conditionswere as given above for the experiments of FIG. 2.

Example 3

[0225] A Multiplexed Reaction with Both Allele 1 and Allele 2: Allele 1is 10 Times More Concentrated than Allele 2.

[0226] The experiment was set up in the following fashion (3 reactiontubes, a triplicate)

[0227] 22 μl of Mastermix

[0228] 13 μl of probes and primers (both the probes were present)

[0229] 5.0 μl of Allele 1

[0230] 0.5 μl of Allele 2

[0231] 9.5 μl of buffer (10 mM Tris-HCl, 11 mM EDTA, pH 8.0)

[0232] The above volumes were added to respective PCR tubes and thereaction mixtures were cycled in the following fashion for 40 cycles.

[0233] Initial Steps:

[0234] The Reaction Mixtures Were Kept at 50° C. for 2 Minutes forOptimal AmpErase UNG Activity. The 10 Minute 95° C. Step was Required toActivate AmpliTaq Gold DNA Polymerase.

[0235] Each of the 40 cycles is performed in the following fashion MeltAnneal/Extend/Cleave 15 seconds 60 seconds 95° C. 60° C.

[0236] The results are shown in FIG. 5. CE separation of a 1:10 mixtureof the 40 cycles products of Alleles 1 and 2. Experimental conditionswere as given for the experiments of FIG. 2.

Example 4 Electroseparation of Label Conjugates on Microfluidic Chip

[0237] Label conjugates comprising fluorescein linked to three differentpeptides, namely, KKAA, KKKA and KKKK were prepared as follows: Theprotected tetrapeptide was prepared on resin using Merrifield reagents.The N-terminus of the last aminoacid was reacted with fluoresceinN-hydroxysuccinimide (Molecular Probes). The peptides were cleaved fromthe resin and purified by high performance liquid chromatography (HPLC).

[0238] The label conjugates prepared as described above and fluoresceinwere combined in an aqueous buffered and were separated and detected inan electrophoresis chip. Detection was 0.5 cm for the injection point onthe anodal side of an electrophoresis channel. FITC-KKKK exhibitednegative charge and FITC-KKKA and FITC-KKKK exhibited positive charge asdetermined by the migration time relative to EOF. The net charge ofFITC-KKKK was greater than +1 and FITC-KKKA and FITC-KKKK migratedelectrophoretically against the EOF. The results are shown in FIG. 6.

Example 5

[0239] Capillar Electrophoresis of CFTR PCR Products with E-Tag Probeson ABI 310

[0240] The following example demonstrates separation in a gel basedcapillary electrophoresis of cleavage of a probe. The conditionsemployed were: Gel: 2.5% LDD30 in 1×TBE with 7M urea; CE: PE ABI 310;Capillary: 47 cm long; 36 cm to window; 75 um ID; Running Buffer: 1×TBE.(LDD30 is a linear copolymer of N,N-diethyl acrylamide andN,N-dimethylacrylamide, 70:30).

[0241] The ABI310 was set up in accordance with the directions of themanufacturer. The parameters used were: Inj Secs 5; Inj kV 2.0; Run kV9.4; Run C 45; Run Time 10 min. To determine the relationship of whereeach probe separated, a spike in system was used. First one digestedprobe was separated and its peak site determined, then a second probewas spiked into the first probe and the two separated. Then, a thirdprobe was spiked in and separated, and so on till the sites of all thesix probes was determined. The single plex PCR runs were first separatedfollowed by separation of the multiplex PCR, which was compared to theS1 digested separation. Type of Probe conc. Sample* probe (nM) vol (μl)10s FAM-T 32 mer 20 10 10s FAM-T dig 20 10 10s FAM-T pcr 80 5 10as HEX-T32 mer 20 10 10as HEX-T dig 20 10 10as HEX-T pcr 80 5 11s HEX-A 28 mer20 10 11s HEX-A dig 20 10 11s HEX-A pcr 80 5 11as TET-C 28 mer 20 1011as TET-C dig 20 10 11as TET-C pcr 80 5 13s FAM-C 23 mer 20 10 13sFAM-C dig 20 10 13s FAM-C pcr 80 5 13as TET-A 23mer 20 10 13as TET-A dig20 10 13as TET-A pcr 80 5 MP10s11s13as pcr 80 5 MP10as11as13a pcr 80 5MP10s10as11s11as13s13as pcr 80 5

[0242] dig —S1 nuclease digestion; pcr−amplification; * the particularsamples are found in the above table, where as exemplary 10 s FAM-Tintends Exon 10 sense, which is referred to as CF7, so that one looks toCF7 for the probe sequence, FAM intends fluorescein, and T is thenucleotide to which the fluorescein is attached. For the other symbols,“as” is the antisense sequence, HEX is hexachlorofluorescein, TET istetrachlorofluorescein, and when more than one exon is indicated, thereaction mixture is multiplexed under the conditions described below

Example 6

[0243] Taq DNA Polymerase exhibits 5′ to 3′ exonuclease activity inwhich hybridized probes on the template DNA are cleaved during PCR. Inthe subject example, sequence specific probes with fluoroscent dyeattached to the 5′ were employed. PCR was performed with these probes ina reaction and then separation performed in a gel based capillaryelectrophoresis to determine the cleavage of the probe.

[0244] Primers, Probes, Mutation Name Location_SNP Mutation SNP CF1 Exon11 R553X C1789T CF2 Exon 19 R1162X C3616T CF4 Exon 3  G85E G386A CF5Exon 4  R117H G482A CF6 Exon 7  R347P G1172C CF7 Exon 10 V520F G1690TCF8 Exon 11 G542X G1756T CF9 Exon 11 G551D G1784A CF10* Exon 11 R560TG1811C CF11* Exon 18 D1152H G3586C CF13* Exon 22 G1349D G4178A NameHyb_probe_length Probe_seq Probe_antisense CF1HYB 26GTGGAGGTCAACGAGCAAGAATTTCT AGAAATTCTTGCTCGTTGACCTCCAG CF2HYB 25AGATGCGATCTGTGAGCCGAGTCTT AAGACTCGGCTCACAGATCGCATCT CF4HYB 32TTCTGGAGATTTATGTTCTATGGAATCTTTTT AAAAAGATTCCATAGAACATAAATCTCCA GAACF5HYB 21 AAGGAGGAACGCTCTATCGGG CGCGATAGAGCGTTCCTCCTT CF6HYB 20ATTGTTCTGCGCATGGCGGT ACCGCCATGCGCAGAACAAT CF7HYB 25ATACAGAAGCGTCATCAAAGCATGC GCATGCTTTGATGACGCTTCTGTAT CF8HYB 29CAATATAGTTCTTGGAGAAGGTGGAATCA TGATTCCACCTTCTCCAAGAACTATATTG CF9HYB 26CTGAGTGGAGGTCAACGAGCAAGAAT ATTCTTGCTCGTTGACCTCCACTCAG CF10HYB* 32TTCCATTTTCTTTTTAGAGCAGTATAAAGA TCTTTGTATACTGCTCTAAAAAGAAAATG GAACE11HYB* 28 AAACTGCAGCATAGATGTGGATAGCTTG CAAGCTATCCACATCTATGCTGGAGTTTCE13HYB* 23 CTAAGCCATGGCCACAAGCAGTT CTGCTTGTGGCCATGGCTTAG Nameproduct_size forward_seq Reverse_seq CF1PF/R 198CCTTTCAAATTCAGATTGAGCATAC TTTACAGCAAATGCTTGCTAGAC CF2PF/R 127TGTGAAATTGTCTGCCAUCTTA GGTTTGGTTGACTTGGTAGGTTTA CF4PF/R 239TCTTTTGCAGAGAATGGGATAGA TGGAGTTGGATTCATCCTTTATATT CF5PF/R 151CCAAAGCAGTACAGCCTCTCTTA CCAAAAATGGCTGGGTGTAG CF6PF/R 137TCTGTGCTTCCCTATGCACTAA CCAAGAGAGTCATACCATGTTTGTA CF7PF/R 146TGGAGCCTTCAGAGGGTAAA TGCTTTGATCACGCTTCTGTA CF8PF/R 198CCTTTCAAATTCAGATTGAGCATAC TTTACAGCAAATGCTTGCTAGAC CF9PF/R 198CCTTTCAAATTCAGATTGAGCATAC TTTACAGCAAATGCTTGCTAGAC F10PF/R 108GACCAGGAAATAGAGAGGAAATGTA CATCTAGGTATCCAAAAGGAGAGTCTA CE11PF/R* 188GAAGGAGAAGGAAGAGTTGGTATTATC CGGTATATAGTTC1TCCTCATGCTATT OF13PF/R* 138TTGGGCTCAGATCTGTGATAG GCAAGATCTTCGCCTTACTG Name Name Tm_prob,Tm_forward, Tm_reverse, oC forward_length, reverse_length CF1HYB CF1PF/R66.83, 60.36, 58.78 25, 23 CF2HYB CF2PF/R 68.65, 59.64, 60.51 23, 24CF4HYB CF4PF/R 64.24, 60.21, 59.2  23, 25 CF5HYB CF5PF/R 65.06, 60.08,60.36 23, 20 CF6HYB CF6PF/R 68.18, 59.9,  59.48 22, 25

[0245] The procedure employed in carrying out the Single-plex PCRreaction was as follows:

[0246] 1. Make up Master Mix 1x 6.5x 13.2 ul 85.8 ul Water 3 ul 19.5 ul25 mM MgCl2 2.5 ul 16.25 ul 10x PCR Buffer 1 ul 6.5 ul 20 ng/ul DNAtemplate 0.2 ul 1.3 ul 25 mM dNTPs 0.3 ul 1.95 ul  5 u/ul Taq Gold (thisis added just prior to start of reaction)

[0247] 2. Aliquot 0.8 ul of 5 uM probe and 4 ul of 10 uM primer set toPCR tubes.

[0248] 3. Primer sets Probe 10s CF10s 10as CF10as 11s CF11s 11as CF11as13s CF13s 13as CF13as

[0249] 4. Aliquot 20.2 ul of the Master Mix to each tube.

[0250] 5. In a PE2400 cycler,

[0251] 96C; 10 MIN

[0252] 35 CYCLES

[0253] 95C; 10 SEC

[0254] 55C; 30 SEC

[0255] 70C; 45 SEC

[0256] 35 CYCLES

[0257] 70C; 10 MIN

[0258] 4C; 24 hours

[0259] 6. After PCR, run the 2.5 ul of each sample on a 2.5% agarosegel.

[0260] 7. EtBr stain the gel, take image with camera equipped UV source.

[0261] Results clearly demonstrated the formation of a uniqueelectrophoretic tag with distinct mobility (Table 1) for each amplifiedsequence.

[0262] Multiplex Amplification of CFTR Fragments with E-Tag Probes

[0263] In this study the reaction involved a plurality of probes in thesame PCR reaction mixture for different snps in CFTR. In the subjectsystem, sequence specific probes with fluorescent dye attached to the 5′terminus of the probe were employed. PCR was performed with these probesand then separation performed in gel based capillary electrophoresis todetermine the cleavage of the probe. The following table indicates thefragment, the mutation reference and the specific nucleotide differenceand number inb the sequence.

[0264] The procedure employed for performing the multiplex amplificationwas as follows:

[0265] Make up Master Mix 1x 2.2x 8 ul 17.6 ul 25 mM MgCl₂ 2.5 ul 5.5 ul10x PCR Buffer 8 ul 17.6 ul 10 ng/ul DNA template .2 ul .44 ul 25 mMdNTPs 1 ul 2.2 ul  5 u/ul Taq Gold (this is added just prior to start ofreaction)

[0266] 8. Aliquot 0.8 ul of each 5 uM probes CF10s, CF11s, CF10as,CF11as, CF13as and 1 ul of each 10 uM primer sets 10s, 11s, 10as, 11as,13as in one PCR tube.

[0267] 9. Aliquot 19.7 ul of the Master Mix to each tube.

[0268] 10. In a PE2400 cycler,

[0269] 96C; 10 MIN

[0270] 40 CYCLES

[0271] 95C; 10 SEC

[0272] 55C; 30 SEC

[0273] 65C; 1 MIN

[0274] 40 CYCLES

[0275] 70C; 10MIN

[0276] 4C; storage

[0277] 11. After PCR, The amplified products were separated as describedin the previous section. The results are shown in FIG. 7. Even in themultiplexed amplification each detection probe gives rise to a uniquee-tag with distinct mobility.

Example 7 Electroseparation of Nine Electrophoretic Tags on MicrofluidicChip

[0278] Label conjugates comprising 9 different fluorescein derivativeslinked to thymine, (Table 1 in Example 6; 1-9): Poly deoxy thymidine(20-mer; with a 5′ thiol group) is reacted with different maleamidefunctionalized fluoresceins. After the reaction the product is ethanolprecipitated. In a reaction of 12 μl in volume, 10 μl of 25 μM oligo,1.0 μl 10×S1 nuclease reaction buffer, 1 μl of S1 nuclease, incubate at37° C. for 30 min followed by 96° C. for 25 min. The digested fragmentsare purified by HPLC.

[0279] The nine different e-tags prepared as described above andfluorescein were combined in an aqueous buffered and were separated anddetected in an electrophoresis chip. Detection was 0.5 cm for theinjection point on the anodal side of an electrophoresis channel. Theresults are shown in FIG. 8.

Example 8

[0280] RT-PCR Conditions:

[0281] Ten ul from a total volume of 25 uls of each mRNA was analyzed ina total volume of 50 uls containing 0.5 uM of each of theoligonucleotide primers, 0.2 mM of each dNTP, 100 nM of each c-taglabeled oligonucleotide probe, 1×RT PCR buffer, 2.5 mM MgCl2, 0.1 U/ulTfl DNA polymerase and 0.1 U/ul AMV Reverse Transcriptase (PromegaAccess, RT-PCR system).

[0282] Reverse Transcription was performed for 45 minutes at 48° C.followed by PCR. (40 thermal cycles of 30 s at 94° C., 1 min at 60° C.and 2 min at 69° C. mRNA was obtained from M. Williams, Genentech Inc.Probe and primer design was performed as described in AnalyticalBiochemistry, 270, 41-49 (1999). Phosphorothioates were attached to 2,3, 4 and 5 phosphate moieties from the 5′ end. Separation was performedas described in the previous section.

[0283]FIG. 9a: Demonstrates the formation of 5 different cleavageproducts in the PCR amplification of ANF with electrophoretic taglabeled at the 5′ end of the sequence detection probe. In the secondexperiment, phosphate group at 2, 3, 4 and 5 position is converted intothiophosphate group. PCR amplification of ANF using thiophospatemodified sequence detection probe yield only one cleavage product.

[0284]FIG. 9b Demonstrates the formation of 3 different cleavageproducts in the PCR amplification of GAPDH with e-tag labeled at the 5′end of the sequence detection probe. In the second experiment, phosphategroup at 2 and 3 position is converted into thiophosphate group. PCRamplification of ANF using thiophospate modified sequence detectionprobe yield only one predominant cleavage product.

[0285] Results clearly demonstrate that for two different genes thatthiophosphates prevent cleavage at multiple sites of detection probes.

[0286] A single detectable entity (a single electrophoretic tag: FIGS.9a and 9 b) is generated as a consequence of amplification reaction.

Example 9

[0287] General Procedure for Synthesis of 6-CarboxyfluoresceinPhosphoramidite Derivatives.

[0288] To a solution of 6-carboxyfluorescein (0.5 g, 1.32 mmol) in drypyridine (5 mL) was added dropwise, isobutyric anhydride (0.55 mL, 3.3mmol). The reaction was allowed to stir at room temperature under anatmosphere of nitrogen for 3 h. After removal of pyridine in vacuo theresidue was redissolved in ethyl acetate (150 mL) and washed with water(150 mL). The organic layer was separated, dried over Na₂SO₄, filtered,and concentrated in vacuo to yield a brownish residue. This material wasdissolved in CH₂Cl₂ (5 mL) after which N-hydroxy succinimide (0.23 g,2.0 mmol) and dicyclohexylcarbodiimide (0.41 g, 1.32 mmol) were added.The reaction was allowed to stir at room temperature for 3 h and thenfiltered through a fritted funnel to remove the white solid, which hadformed. To the filtrate was added aminoethanol (0.12 mL, 2.0 mmol)dissolved in 1 mL of CH₂Cl₂. After 3 h the reaction was again filteredto remove a solid which had formed and then diluted with additionalCH₂Cl₂ (50 mL). The solution was washed with water (150 mL) and thenseparated. The organic layer was dried over Na₂SO₄, filtered, andconcentrated in vacuo to yield a white foam (0.7 g, 95%, 3 steps). ¹HNMR: (DMSO) δ 8.68 (t, 1H), 8.21 (d, 1H), 8.14 (d, 1H), 7.83 (s, 1H),7.31 (s, 2H), 6.95 (s, 4H), 4.69 (t, 1H), 3.45 (q, 2H), 3.25 (q, 2H),2.84 (h, 2H), 1.25 (d, 12H). Mass (LR FAB⁺) calculated for C₃₁H₂₉NO₉(M+H⁺) 559.2, found: 560.

[0289] It is evident from the above results that the subject inventionprovides an accurate, efficient and sensitive process, as well ascompositions for use in the process, to perform multiplexed reactions.The protocols provide for great flexibility in the manner in whichdeterminations are carried out and maybe applied to a wide variety ofsituations involving hpatens, antigens, nucleic acids, cells, etc.,where one may simultaneously perform a number of determinations on asingle or plurality of samples and interrogate the samples for aplurality of events. The events may vary from differences in nucleicacid sequence to proteomics to enzyme activities. The results of thedetermination are readily read in a simple manner using electrophoresisor mass spectrometry. Systems are provided where the entire process,after addition of the sample and reagents, maybe performed under thecontrol of a data processor with the results automatically recorded.

[0290] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0291] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1 53 1 16 DNA Artificial Sequence Probe 1 tcaccacatc ccagtg 16 2 16 DNAArtificial Sequence Probe 2 gagggaggtt tggctg 16 3 22 DNA ArtificialSequence Probe 3 ccagcaacca atgatgcccg tt 22 4 22 DNA ArtificialSequence Probe 4 ccagcaagca ctgatgcctg tt 22 5 4 PRT Artificial SequenceElectrophoretic mobility modifier 5 Lys Lys Ala Ala 6 4 PRT ArtificialSequence Electrophoretic mobility modifier 6 Lys Lys Lys Ala 7 4 PRTArtificial Sequence Electrophoretic mobility modifier 7 Lys Lys Lys Lys8 26 DNA Artificial Sequence Probe 8 gtggaggtca acgagcaaga atttct 26 926 DNA Artificial Sequence Probe 9 agaaattctt gctcgttgac ctccac 26 10 25DNA Artificial Sequence Probe 10 agatgcgatc tgtgagccga gtctt 25 11 25DNA Artificial Sequence Probe 11 aagactcggc tcacagatcg catct 25 12 32DNA Artificial Sequence Probe 12 ttctggagat ttatgttcta tggaatcttt tt 3213 32 DNA Artificial Sequence Probe 13 aaaaagattc catagaacat aaatctccagaa 32 14 21 DNA Artificial Sequence Probe 14 aaggaggaac gctctatcgc g 2115 21 DNA Artificial Sequence Probe 15 cgcgatagag cgttcctcct t 21 16 20DNA Artificial Sequence Probe 16 attgttctgc gcatggcggt 20 17 20 DNAArtificial Sequence Probe 17 accgccatgc gcagaacaat 20 18 25 DNAArtificial Sequence Probe 18 atacagaagc gtcatcaaag catgc 25 19 25 DNAArtificial Sequence Probe 19 gcatgctttg atgacgcttc tgtat 25 20 29 DNAArtificial Sequence Probe 20 caatatagtt cttggagaag gtggaatca 29 21 29DNA Artificial Sequence Probe 21 tgattccacc ttctccaaga actatattg 29 2226 DNA Artificial Sequence Probe 22 ctgagtggag gtcaacgagc aagaat 26 2326 DNA Artificial Sequence Probe 23 attcttgctc gttgacctcc actcag 26 2432 DNA Artificial Sequence Probe 24 ttccattttc tttttagagc agtatacaaa ga32 25 32 DNA Artificial Sequence Probe 25 tctttgtata ctgctctaaaaagaaaatgg aa 32 26 28 DNA Artificial Sequence Probe 26 aaactccagcatagatgtgg atagcttg 28 27 28 DNA Artificial Sequence Probe 27 caagctatccacatctatgc tggagttt 28 28 23 DNA Artificial Sequence Probe 28 ctaagccatggccacaagca gtt 23 29 23 DNA Artificial Sequence Probe 29 aactgcttgtggccatggct tag 23 30 25 DNA Artificial Sequence Primer 30 cctttcaaattcagattgag catac 25 31 23 DNA Artificial Sequence Primer 31 tttacagcaaatgcttgcta gac 23 32 23 DNA Artificial Sequence Primer 32 tgtgaaattgtctgccattc tta 23 33 24 DNA Artificial Sequence Primer 33 ggtttggttgacttggtagg ttta 24 34 23 DNA Artificial Sequence Primer 34 tcttttgcagagaatgggat aga 23 35 25 DNA Artificial Sequence Primer 35 tggagttggattcatccttt atatt 25 36 23 DNA Artificial Sequence Primer 36 ccaaagcagtacagcctctc tta 23 37 20 DNA Artificial Sequence Primer 37 ccaaaaatggctgggtgtag 20 38 22 DNA Artificial Sequence Primer 38 tctgtgcttccctatgcact aa 22 39 25 DNA Artificial Sequence Primer 39 ccaagagagtcataccatgt ttgta 25 40 20 DNA Artificial Sequence Primer 40 tggagccttcagagggtaaa 20 41 21 DNA Artificial Sequence Primer 41 tgctttgatgacgcttctgt a 21 42 25 DNA Artificial Sequence Primer 42 cctttcaaattcagattgag catac 25 43 23 DNA Artificial Sequence Primer 43 tttacagcaaatgcttgcta gac 23 44 25 DNA Artificial Sequence Primer 44 cctttcaaattcagattgag catac 25 45 23 DNA Artificial Sequence Primer 45 tttacagcaaatgcttgcta gac 23 46 25 DNA Artificial Sequence Primer 46 gaccaggaaatagagaggaa atgta 25 47 26 DNA Artificial Sequence Primer 47 catctaggtatccaaaggag agtcta 26 48 27 DNA Artificial Sequence Primer 48 gaaggagaaggaagagttgg tattatc 27 49 26 DNA Artificial Sequence Primer 49 cggtatatagttcttctcat gctatt 26 50 21 DNA Artificial Sequence Primer 50 ttgggctcagatctgtgata g 21 51 20 DNA Artificial Sequence Primer 51 gcaagatcttcgccttactg 20 52 21 DNA Artificial Sequence Probe 52 cagcaaccattgatgcccgt t 21 53 21 DNA Artificial Sequence Probe 53 cagcaagcactgatgcctgt t 21

What is claimed is:
 1. A system for the simultaneous multiplexed determination of a plurality of events employing electrophoresis to distinguish the events, comprising an electrophoretic device for electrophoretic separation and detection, a container containing a first set of first agents comprising differing mobility regions defining differing target-binding regions and a second reagent comprising at least one active agent under conditions where said active agent modifies members of said first set bound to a target resulting in a change of electrophoretic mobility of said first agents bound to target to provide a modified member retaining said mobility region, and transfer of said at least one modified member to said electrophoretic device for separation and detection of said at least one modified member, with the proviso that when said first and second reagents comprise oligonucleotides, said mobility region is other than an oligomer.
 2. A system for the simultaneous multiplexed determination of a plurality of events employing electrophoresis to distinguish the events, employing e-tags comprising a region having two functionalities, a first functionality for binding to or bound to a detectable label and a second functionality bonded to a target-binding region, said system comprising a vessel containing a plurality of e-tag moieties with each e-tag bonded to a different target-binding region, and a sample, whereby the presence in the sample of a target for each of said e-tag moieties results in a modification of said c-tag to produce modified e-tags with a change in mobility of said e-tag, and an electrophoresis device for separating and detecting said modified e-tags, means for moving said modified e-tags to said electrophoresis device and a data processor for receiving and processing data from said electrophoresis device, with the proviso that in the event that said first functionality is not bound to said detectable label, said system further comprising joining said modified e-tag to said detectable label and when said first and second reagents comprise oligonucleotides, said mobility region is other than an oligomer
 3. A system according to claim 1, wherein said target-binding region is a polynucleotide.
 4. A system according to claim 1, wherein said target-binding region is a poly(amino acid).
 5. A system according to claim 1, wherein said second functionality or said target-binding region is cleavable and said system further comprises cleavage of said second functionality or said target-binding region, respectively.
 6. A system according to claim 1, wherein said electrophoresis device is a capillary electrophoresis device.
 7. A system according to claim 5, wherein said capillary electrophoresis device is a microfluidic device.
 8. A method for performing multiplexed determinations in a nucleic acid sample, employing a reagent having a plurality of first members, each first member having an oligonucleotide sequence homologous to a target sequence and a non-oligomeric e-tag bonded to a nucleotide, and a second reagent having a nucleic acid sequence homologous to a target sequence proximal to said first member homologous sequence, with the proviso that when said first member comprises a DNA sequence interrupted by an RNA sequence a ribonuclease specific for a chimeric double stranded nucleic acid is substituted for said second reagent, said method comprising: combining a target nucleic acid sample with said first and second reagents under conditions where said first and second reagents comprising nucleic acid sequences bind to homologous target sequences and said first and/or second reagent is modified to change the mobility of said e-tag by joining any first reagent and second reagent bound to target nucleic acid, releasing said e-tag from at least a portion of said sequence of said first reagent or cleaving said RNA interrupting said DNA to produce modified e-tags; and separating said modified e-tags by electrophoresis, whereby the presence of target nucleic acid is determined.
 9. A method according to claim 8, wherein at least one link in said nucleic acid sequence of said first reagent is resistant to nuclease cleavage.
 10. A method according to claim 8, wherein a partitioning ligand is bonded to said first nucleic acid sequence where upon release of said e-tag, said partitioning ligand is retained with the remaining portion of said first sequence, and further including the step after said combining, of binding said partitioning ligand with a receptor to diminish any interference in said separating from components of said first and second reagents retaining said ligand.
 11. A method according to claim 8, wherein said e-tag mobility variation is based at least in part on variation in substitution of a fluorescent label bonded to said e-tag.
 12. A method according to claim 8, wherein said e-tag mobility variation is based at least in part on differences in the length of an alkylene or aralkylene group or combination of alkylene or aralkylene group joined by a polar group.
 13. A method for detecting at least one target nucleic acid sequence in a nucleic acid sample, said method comprising: combining under bond cleavage conditions: a reagent system capable of cleaving a cleavable bond of an e-tag linked target-binding sequence, said nucleic acid sample and a reagent pair consisting of a primer and said e-tag linked target-binding sequence, each reagent pair having sequences homologous for each nucleic acid sequence to be determined, wherein each said primer specifically binds to said target nucleic acid and said target-binding sequence binds to said target nucleic acid downstream from said primer, wherein each said target-binding sequence is characterized by being linked to a non-oligomeric e-tag specific for each said nucleic acid sequence; executing at least one cycle of cleavage of said cleavable bond, whereby said e-tag is released substantially free of said target-binding sequence; separating released e-tags into individual fractions; and detecting said e-tag fractions, whereby the presence in said target nucleic acid sample of said at least one nucleic acid sequence is detected; with the proviso that, when separation is performed solely by means of differences in mass, the e-tags that are separated all have the same number of nucleotides bonded to the e-tag.
 14. A method according to claim 13, wherein said bond cleavage conditions comprise a 5′-3′-nuclease.
 15. A method according to claim 13, wherein said bond cleavage conditions comprise a reagent on said primer causing cleavage of said bond.
 16. A method according to claim 15, wherein said reagent is an enzyme that produces singlet oxygen or hydrogen peroxide and said cleavable bond is oxidatively cleaved.
 17. A method according to claim 13 wherein at least one link in said nucleic acid sequence of said target-binding sequence is resistant to nuclease cleavage.
 18. A method according to claim 13, wherein a partitioning ligand is bonded to said e-tag linked target binding sequence where upon release of said e-tag, said partitioning ligand is retained with the remaining portion of said target-binding sequence, and further including the step after said combining, binding said partitioning ligand with a receptor to diminish any interference in said separating from ligand containing components.
 19. A method according to claim 13, wherein said at least one target nucleic acid sequence comprises a snp and said bond cleavage conditions comprise a 5′-3′ nuclease, wherein either said target-binding sequence or said primer includes a nucleotide complementary to said snp.
 20. A method for performing a plurality of simultaneous determinations in a vessel in relation to a plurality of targets, each target having at least one epitope, employing a first composition with different e-tags specific for each of said targets linked to a target-binding region that comprises a reciprocal binding member to said epitopes, or said first composition is different candidate enzyme substrates and said e-tags are specific for each enzyme substrate, said method comprising: combining said first composition with a second composition, wherein said second composition is suspected of containing at least one target or contains at least one enzyme for said candidate substrates, and additional reagents, so that binding of said target-binding sequence to a target results in a change in the mass or electrophoretic mobility of said e-tag linked target-binding sequence to produce a product; separating said products into discrete packets for identification; whereby said determinations are made as to each of said e-tags.
 21. A method according to claim 20, wherein said targets have a plurality of epitopes and said additional reagents comprise a receptor for said targets bound to a support and including the additional steps of separating said e-tags bound to said support from unbound e-tags and cleaving said cleavable bond releasing said e-tags from at least a portion of said reagent region.
 22. A method according to claim 20, wherein said targets have a plurality of epitopes and said second composition is said enzyme, wherein said enzyme produces singlet oxygen or hydrogen peroxide.
 23. A library comprising a plurality of non-oligomeric e-tags, said e-tags having a molecular weight in the range of about 150 to 5,000, each e-tag, with the entities to which it is attached, providing a different mobility in electrophoresis, wherein at least 5 of the e-tags comprise: a mobility-identifying region comprising at least one negative or positive charge, a target-binding region comprising at least one nucleotide or nucleotide analog, an amino acid or poly(amino acid), an enzyme substrate or a first functionality for bonding to any of them; a detectable label or a second functionality for binding to a detectable label, wherein when said mobility-modifying region is joined to or for joining to said poly(amino acid), said first functionality is a cleavable bond.
 24. A library according to claim 23, wherein said target-binding region comprises a nucleotide or nucleotide analog.
 25. A library according to claim 23, wherein said target-binding region is an oligonucleotide, wherein at least one linkage between nucleotides is nuclease resistant.
 26. A library according to claim 23, wherein said second functionality is for bonding to a poly(amino acid). 